JP7373588B2 - Anti-CTLA-4 antibody and its use - Google Patents

Description

The present invention relates to anti-CTLA-4 antibodies and methods of using the same.

Cells that have mutated in vivo due to genetic mutations are monitored and eliminated by the immune surveillance system. On the other hand, the persistence of an excessive immune response can also be harmful to oneself, such as damage to normal tissues due to autoimmunity. Therefore, the immune system is equipped with a negative feedback mechanism (immune checkpoint) for suppressing the immune reaction once activated (see, for example, Non-Patent Document 1). Immune checkpoints are thought to play an important role in maintaining homeostasis in the immune system. On the other hand, it has become clear that some tumors use immune checkpoints to evade the immune system. Currently, immunity is mediated by major immune checkpoint molecules such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1). Research on the inhibitory function is widely underway.
CTLA-4 is a glycoprotein belonging to the immunoglobulin superfamily whose gene was cloned from a cDNA library of a mouse-derived killer T cell clone in 1987 (see, for example, Non-Patent Document 2). It is known that T cell immune responses are suppressed through CTLA-4. Based on the idea that suppressing the function of CTLA-4 and promoting T cell activation would lead to cancer regression, in 1996, administration of anti-CTLA-4 antibodies to tumor-bearing mice had a tumor regression effect. It has been reported that this was observed (see, for example, Non-Patent Document 3). Since 2000, the effectiveness of anti-CTLA-4 antibodies in humans has been evaluated, and in 2011, anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the U.S. Food and Drug Administration (FDA) for the first time in the world. The drug was approved as an immune-activating antibody drug. In addition to ipilimumab, many anti-CTLA-4 monoclonal antibodies have been produced (see, for example, Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4), and attempts are being made to develop them as pharmaceuticals. These drugs, which inhibit immune checkpoints to release their immunosuppressive mechanisms and, as a result, increase immune activity, are called immune checkpoint inhibitors.
On the other hand, it has long been known that some T cells have immunosuppressive functions, and in 1995 they were identified as CD25-positive and CD4-positive T cells, and they were classified as regulatory T cells. (For example, see Non-Patent Document 4). In 2003, the Foxp3 gene, a master gene that is specifically expressed in regulatory T cells and controls their development and function, was identified. Foxp3 regulates the expression of various immune response-related genes as a transcription factor. Foxp3 is particularly involved in the constitutive expression of CTLA-4 in regulatory T cells, which is thought to play an important role in the immunosuppressive function of regulatory T cells (e.g., (See Patent Document 5).
The infiltration of regulatory T cells into tumor tissue is thought to lead to the weakening or inhibition of immune surveillance mechanisms against tumors. In fact, it has been revealed that regulatory T cells increase in many human cancers (see, for example, Non-Patent Document 6), and regulatory T cells infiltrate locally into tumors in cancer patients. It has been reported that it can be a factor in poor prognosis. Conversely, if regulatory T cells can be removed or reduced from tumor tissue, it is expected to lead to enhanced anti-tumor immunity. Currently, the development of cancer immunotherapy targeting regulatory T cells is being actively pursued.
Administration of ipilimumab, an anti-CTLA-4 antibody, enhances antitumor immunity, but on the other hand, it has been reported that autoimmune diseases develop due to the systemic enhancement of immune activity. In one clinical trial, 60% of patients treated with ipilimumab experienced adverse events, many of which were autoimmune diseases related to the skin or gastrointestinal tract. In other clinical trials, it has been reported that approximately half of patients treated with ipilimumab developed similar autoimmune diseases. To suppress these side effects, patients receiving ipilimumab are sometimes given immunosuppressants. There is a desire to develop new drugs that can maintain antitumor immune responses while suppressing the side effects of these immune checkpoint inhibitors.
Antibody-dependent cytotoxic activity (ADCC), complement-dependent cytotoxic activity (CDC), and antibody-dependent cytophagic activity (ADCP), which are cytotoxic effector functions of IgG antibodies, are used to obtain antitumor effects from antibodies. It is attracting attention as a promising means (for example, see Non-Patent Document 7 and Non-Patent Document 8). These effector functions are induced when the Fc region of an IgG antibody binds to antibody receptors (FcγR) or various complement components present on the surface of effector cells such as natural killer cells and macrophages. A large number of studies have been conducted on Fc region mutants to date, and mutants with various properties such as higher FcγR binding activity than the wild type have been obtained (for example, Patent Document 5, Patent Document 6, Non-Patent Document 6, (See Document 9 and Non-Patent Document 10). Furthermore, it has been reported that the Fc region of an antibody binds to FcγR in a 1:1 ratio and recognizes FcγR asymmetrically at the lower hinge and CH2 regions (see, for example, Non-Patent Document 11). Based on this, a method has also been reported to optimize the interaction with FcγR by making asymmetric Fc region variants by making different modifications to the two polypeptide chains that make up the Fc region of antibodies. (For example, see Patent Document 7, Patent Document 8, Patent Document 9, and Patent Document 10).
When a therapeutic antibody is administered in vivo, it is desirable that the target antigen is specifically expressed only in the lesion site, but in many cases, the same antigen is also expressed in normal tissue, which is a non-lesion site. This can lead to undesirable side effects from a therapeutic point of view. For example, while an antibody against a tumor antigen can exert cytotoxic activity against tumor cells through ADCC or the like, if the same antigen is also expressed in normal tissue, it may also damage normal cells. In order to solve the above-mentioned problems, we focused on the phenomenon in which specific compounds are present in large amounts in target tissues (e.g., tumor tissues), and developed antigen binding in which the antigen-binding activity changes depending on the concentration of such compounds. Techniques for creating molecules have been developed (see, for example, Patent Document 11).

WO 2000/037504 WO 2001/014424 WO 2012/120125 WO 2016/196237 WO 2000/042072 WO 2006/019447 WO 2012/058768 WO 2012/125850 WO 2013/002362 WO 2014/104165 WO 2013/180200

Pardoll, Nat Rev Cancer (2012) 12: 252-264 Brunet et al., Nature (1987) 328: 267-270 Leach et al., Science (1996) 271: 1734-1736 Sakaguchi et al., J Immunol (1995) 155: 1151-1164 Takahashi et al., J Exp Med (2000) 192: 303-310 Nishikawa & Sakaguchi, Int J Cancer (2010) 127: 759-767 Clynes et al., Proc Natl Acad Sci U S A (1998) 95: 652-656 Clynes et al., Nat Med (2000) 6: 443-446 Lazar et al., Proc Natl Acad Sci U S A (2006) 103: 4005-4010 Chu et al., Mol Immunol (2008) 45: 3926-3933 Radaev et al., J Biol Chem (2001) 276: 16469-16477

The present invention provides anti-CTLA-4 antibodies and methods for their use. The invention also provides polypeptides containing variant Fc regions and methods of producing them.

More specifically, the present invention provides the following [1] to [47].
[1] An anti-CTLA-4 antibody having CTLA-4 binding activity dependent on the concentration of an adenosine-containing compound, and having at least one characteristic selected from the following (a) to (i):
(a) the binding activity in the presence of 100 μM of the adenosine-containing compound is more than twice as high as the binding activity in the absence of the adenosine-containing compound;
(b) the KD value in the presence of 100 μM adenosine-containing compound is 5×10 ⁻⁷ M or less;
(c) the KD value in the absence of an adenosine-containing compound is 1×10 ^-6 M or more;
(d) forming a ternary complex with an adenosine-containing compound and CTLA-4;
(e) binds to the region from amino acid 97 to amino acid 106 of human CTLA-4 (extracellular domain, SEQ ID NO: 28);
(f) competes with ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11) for binding to CTLA-4;
(g) binds to the same epitope bound by ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11);
(h) exhibits cytotoxic activity against CTLA-4 expressing cells, and
(i) Binds to human and mouse-derived CTLA-4.
[2] The antibody according to [1], which is a monoclonal antibody.
[3] The antibody according to [1] or [2], which is a human antibody, a humanized antibody, or a chimeric antibody.
[4] The antibody according to any one of [1] to [3], which is an antibody fragment that binds to CTLA-4.
[5] (a) HVR-H1 (SEQ ID NO: 223) comprising the amino acid sequence SX ₁ TMN, where X ₁ is H, A, R, or K, (b) amino acid sequence SISX ₁ X ₂ SX ₃ YIYYAX _{4 HVR - H2 ( array _} No.: 224), and (c) any one of [1] to [4] containing HVR-H3 (SEQ ID NO: 225), where X ₁ is K or A, and (c) contains the amino acid sequence YGX ₁ REDMLWVFDY, and X 1 is K or A. Antibodies described.
[6] (a) Amino acid sequence containing X ₁ GX ₂ STX ₃ VGDYX _{4 X 5} VX ₆ , where X ₁ is T, D, Q, or E, X ₂ is T or P, HVR _- L1 (SEQ ID NO _: 226) where ₄ is N or T, X ₅ is Y or W, and _{X 6} is S _or H, (b) contains _the amino acid sequence HVR-L2 (SEQ ID NO: 227), where E, F, or Y, X ₂ is S or I, X ₃ is K or S, X ₄ is S, E, or _K; The antibody according to [5], which further comprises HVR-L3 (SEQ ID NO: 228), which comprises TYAAPLGPX ₂ , X ₁ is S or Q, and X ₂ is M or T.
[7] Heavy chain variable domain FR1 comprising the amino acid sequence of any one of SEQ ID NO: 229 to 232, FR2 comprising the amino acid sequence of SEQ ID NO: 233, FR3 comprising the amino acid sequence of SEQ ID NO: 234, and SEQ ID NO: The antibody according to [5], further comprising FR4 comprising an amino acid sequence of 235.
[8] Light chain variable domain FR1 comprising any one amino acid sequence of SEQ ID NO: 236-238, FR2 comprising any one amino acid sequence of SEQ ID NO: 240-241, any one of SEQ ID NO: 242-244 The antibody according to [6], further comprising FR3 comprising one amino acid sequence and FR4 comprising any one amino acid sequence of SEQ ID NO: 245 to 246.
[9] (a) a VH sequence having at least 95% sequence identity with any one amino acid sequence of SEQ ID NOs: 83-86, 98, 135-141; (b) SEQ ID NOs: 88-95, 97; (c) a VL sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 83-86, 98, 135-141; and a VL sequence having an amino acid sequence of any one of SEQ ID NOs: 88-95, 97, 99, 134, 144-149, according to any one of [1] to [4]. antibodies.
[10] The antibody according to any one of [1] to [3] and [5] to [9], which is a full-length IgG1 antibody.
[11] A mutant Fc region in which the Fc region contains an amino acid modification, and the mutant Fc region is more effective than a natural Fc region in at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa. The antibody according to [10], which has enhanced binding activity to.
[12] An isolated nucleic acid encoding the antibody according to any one of [1] to [11].
[13] A host cell comprising the nucleic acid according to [12].
[14] A method for producing an antibody, the method comprising culturing the host cell according to [13] so as to produce the antibody.
[15] A pharmaceutical preparation comprising the antibody according to any one of [1] to [11] and a pharmaceutically acceptable carrier.
[16] The pharmaceutical preparation according to [15], wherein the antibody is an immunoconjugate.
[17] The pharmaceutical preparation according to [15] or [16], which is used in combination with at least one selected from the group consisting of an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, and a chemotherapeutic agent.
[18] The pharmaceutical formulation according to any one of [15] to [17] for use in tumor treatment.
[19] The pharmaceutical preparation according to [18], wherein the tumor is a solid tumor infiltrated with regulatory T (Treg) cells.
[20] The pharmaceutical formulation according to any one of [15] to [17] for use in cell injury.
[21] The pharmaceutical formulation according to any one of [15] to [17] for use in damaging Treg cells.
[22] The pharmaceutical preparation according to [20], wherein the cell damage is due to ADCC activity, CDC activity, or ADCP activity.
[23] The pharmaceutical preparation according to [20] or [21], in which immunity is activated by Treg cell damage.
[24] The pharmaceutical formulation according to any one of [15] to [17] for use in immune activation.
[25] The pharmaceutical preparation according to [24], wherein the immune activation is T cell activation.
[26] The pharmaceutical preparation according to [24] or [25], which activates immunity in tumor tissue.
[27] The pharmaceutical according to any one of [24] to [26], which has a lower level of immune activation in non-tumor tissues than a pharmaceutical preparation containing a control anti-CTLA-4 antibody. target formulation.
[28] The pharmaceutical preparation according to any one of [24] to [27], which has a lower level of side effects than a control pharmaceutical preparation containing an anti-CTLA-4 antibody.
[29] Any one of [24] to [28], wherein the anti-CTLA-4 antibody used as a control is an anti-CTLA-4 antibody that does not have CTLA-4 binding activity that is dependent on the concentration of the adenosine-containing compound. Pharmaceutical preparations as described in Section.
[30] The pharmaceutical preparation according to [29], wherein the side effect is an autoimmune disease.
[31] The pharmaceutical preparation according to any one of [18], [19], and [26] to [30], wherein the tumor is breast cancer or liver cancer.
[32] A polypeptide comprising a mutant Fc region containing an amino acid modification in the parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains an amino acid modification at the following positions: , polypeptide:
(i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 in the first polypeptide of the parent Fc region, and
(ii) positions 236, 250, 270, 298, 307, 326, and 334, expressed in EU numbering, in the second polypeptide of the parent Fc region;
[33] The polypeptide according to [32], wherein the mutant Fc region further comprises an amino acid modification at position 332, expressed in EU numbering, in the first polypeptide of the parent Fc region.
[34] The polypeptide according to [32] or [33], wherein the mutant Fc region further comprises an amino acid modification at position 332, expressed in EU numbering, in the second polypeptide of the parent Fc region.
[35] The polypeptide according to any one of [32] to [34], wherein the mutant Fc region further comprises an amino acid modification at position 330 expressed in EU numbering in the second polypeptide of the parent Fc region. peptide.
[36] The polypeptide according to any one of [32] to [35], wherein the mutant Fc region further comprises an amino acid modification at position 356 expressed in EU numbering in the first polypeptide of the parent Fc region. peptide.
[37] The polypeptide according to any one of [32] to [36], wherein the mutant Fc region further comprises an amino acid modification at position 366 expressed in EU numbering in the first polypeptide of the parent Fc region. peptide.
[38] The polypeptide according to any one of [32] to [37], wherein the mutant Fc region further comprises an amino acid modification at position 439, expressed in EU numbering, in the second polypeptide of the parent Fc region. peptide.
[39] Any one of [32] to [38], wherein the mutant Fc region further comprises amino acid modifications at positions 366, 368, and 407 expressed in EU numbering in the second polypeptide of the parent Fc region. The polypeptide described in Section.
[40] The polypeptide according to any one of [32] to [39], which contains at least one amino acid modification selected from the amino acid modifications described below:
(i) Tyr or Phe at position 234 in EU numbering, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, position 268 in the first polypeptide of the parent Fc region; Asp at position 270, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Glu at position 332, Cys at position 349, Lys at position 356, and Trp at position 366, and
(ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, expressed in EU numbering in the second polypeptide of the parent Fc region; Met, Lys at position 330, Asp or Glu at position 332, Glu at position 334, Cys at position 356, Ser at position 366, Ala at position 368, Val at position 407, and Glu at position 439.
[41] From [32], wherein the mutant Fc region further comprises any of the following amino acid modifications in (a) to (d) in the first polypeptide and/or second polypeptide of the parent Fc region: The polypeptide according to any one of [40]:
(a) Ala at position 434 in EU numbering;
(b) Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, expressed in EU numbering;
(c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, expressed in EU numbering;
(d) Leu at position 428, Ala at position 434, Arg at position 438, Glu at position 440 expressed in EU numbering.
[42] The binding activity for at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa is enhanced in the mutant Fc region compared to the parent Fc region, [32] to [41] ] The polypeptide according to any one of the above.
[43] The polypeptide according to [42], wherein the mutant Fc region has enhanced binding activity for FcγRIIa and FcγRIIIa as compared to the parent Fc region.
[44] Any one of [32] to [43], wherein the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors is improved in the mutant Fc region compared to the parent Fc region. The polypeptide described in.
[45] The polypeptide according to [44], wherein the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, and FcγRIIIa, and the inhibitory Fcγ receptor is FcγRIIb.
[46] The polypeptide according to any one of [32] to [45], wherein the polypeptide comprising a mutant Fc region is an antibody.
[47] A method for producing a polypeptide containing a mutant Fc region, the method comprising the step of introducing an amino acid modification into a parent Fc region, the parent Fc region being composed of two polypeptide chains, and at the following positions: Methods by which amino acid modifications are introduced:
(i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 in the first polypeptide of the parent Fc region, and
(ii) positions 236, 250, 270, 298, 307, 326, and 334, expressed in EU numbering, in the second polypeptide of the parent Fc region;

FIG. 1 is a diagram showing the binding activity of anti-CTLA-4 antibody ABAM004 to CTLA-4 depending on ATP, ADP, or AMP concentration, as described in Examples 1-9. FIG. 2 is a diagram showing the binding activity of ABAM004, an anti-CTLA-4 antibody, to CTLA-4 expressing cells depending on the AMP concentration, as described in Example 1-10. FIG. 3 is a diagram showing the ADCC activity of ABAM004, an anti-CTLA-4 antibody, against CTLA-4-expressing cells in the presence and absence of AMP, as described in Example 1-11. FIG. 4 is a diagram showing the mode of binding of ABAM004 Fab fragment and AMP, as described in Example 2-13. In the figure, the heavy chain of the antibody is shown in black, the light chain is shown in gray, and AMP is shown in a ball-and-stick model. Amino acid residues that interact with AMP are shown using a stick model. The dashed line and its numerical value indicate the distance (Å) between each amino acid residue and AMP. FIG. 5 is a diagram showing the binding mode of ABAM004 Fab fragment, AMP, and human CTLA4 (hCTLA4), as described in Example 2-14. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, hCTLA4 in white, and AMP shown in a ball-and-stick model. Amino acid residues of hCTLA4 containing one or more non-hydrogen atoms located within 4.2 Å from any part of the antibody or AMP are defined as epitopes and are shown in a stick model. FIG. 6 is a mapping of the epitope of ABAM004 Fab fragment into the amino acid sequence of hCTLA4, as described in Examples 2-14. In the figure, the amino acid residues shown in black indicate the amino acid residues of hCTLA4 that contain one or more non-hydrogen atoms located within 4.2 Å from either part of ABAM004 or AMP in the crystal structure. Amino acid residues shown in gray are disordered in the crystal structure, and therefore a model could not be constructed. Figure 7 shows the structure of the antibody and AMP extracted from the crystal structures of the complex of ABAM004 Fab fragment alone and AMP, and the three-way complex with AMP and CTLA4, as described in Example 2-15. This is a superimposed diagram. In the figure, the heavy chain of the antibody is shown in black, the light chain is shown in gray, and AMP is shown in a ball-and-stick model. The thin line shows the structure of the ABAM004 Fab fragment alone, the medium thick line shows the structure of the binary complex with AMP, and the thick line shows the structure of the ternary complex. FIG. 8 is a diagram showing the binding activity of anti-CTLA-4 antibody ABAM004 and its variant 04H0150/04L0072 to CTLA-4 depending on ATP, ADP, or AMP concentration, as described in Example 3-2. It is. In the diagram, WT indicates ABAM004, and H150L072 indicates 04H0150/04L0072. FIG. 9 is a diagram showing the ATP concentration-dependent neutralizing activity of anti-CTLA-4 antibody SW1077 against CTLA-4, as described in Example 3-6. FIG. 10 is a diagram showing the antitumor effect of anti-CTLA-4 antibody mNS-mFa55 (control antibody) in a mouse model transplanted with FM3A cell line, as described in Example 3-7-4. Antibodies were administered via the tail vein at 0.01 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 1 mg/kg, 10 mg/kg, 30 mg/kg, and 100 mg/kg. Each point represents the average tumor volume of n=4 in a group. Figure 11 is a diagram showing the antitumor effect of anti-CTLA-4 antibody SW1208-mFa55 (switch antibody) in a mouse model transplanted with FM3A cell line, as described in Example 3-7-4. It is. Antibodies were administered via the tail vein at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg. Each point represents the average tumor volume of n=4 in a group. Figure 12 shows the administration of anti-CTLA-4 antibodies mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody) in a mouse model transplanted with FM3A cell line, as described in Example 3-7-7. FIG. 3 is a diagram showing changes in the proportion of effector Treg cells within tumors. mNS-mFa55 was administered via the tail vein at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and SW1208-mFa55 was administered at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, It was administered through the tail vein at 100 mg/kg and 500 mg/kg. On the 6th day after administration, tumor specimens were collected and FACS analysis was performed to evaluate the increase or decrease in effector Tregs. The vertical axis is the ratio of effector Tregs (CD4 ⁺ FoxP3 ⁺ KLRG1 ⁺ ) to CD45 ⁺ cells. The average value of n=3 is shown. Figure 13 shows the administration of anti-CTLA-4 antibodies mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody) in a mouse model transplanted with FM3A cell line, as described in Example 3-7-8. FIG. 3 is a diagram showing changes in the percentage of activated helper T cells in the spleen of patients. mNS-mFa55 was administered via the tail vein at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and SW1208-mFa55 was administered at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, It was administered through the tail vein at 100 mg/kg and 500 mg/kg. On the 6th day after administration, the spleen was collected, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis is the ratio of activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) to CD45 ⁺ cells. The average value of n=3 is shown. Figure 14 shows the antitumor effect of anti-CTLA-4 antibody SW1389-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Example 4-3-5. It is a diagram. Antibodies were administered via the tail vein at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 100 mg/kg. Each point represents the average tumor volume of n=4 in a group. Figure 15 shows the antitumor effect of anti-CTLA-4 antibody hNS-mFa55 (control antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Example 4-3-5. It is a diagram. Antibodies were administered via the tail vein at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 30 mg/kg. Each point represents the average tumor volume of n=4 in a group. Figure 16 shows anti-CTLA-4 antibody hNS-mFa55 (control antibody) and SW1389-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Example 4-3-8. FIG. 3 is a diagram showing changes in the percentage of effector Treg cells within a tumor upon administration of the antibody. hNS-mFa55 at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 30 mg/kg, SW1389-mFa55 at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, 500 mg /kg was administered via the tail vein. On the 6th day after administration, tumor specimens were collected and FACS analysis was performed to evaluate the increase or decrease in effector Tregs. The vertical axis is the ratio of effector Tregs (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1 ⁺ ) to CD45 ⁺ cells. The average value of n=3 is shown. Figure 17 shows anti-CTLA-4 antibody hNS-mFa55 (control antibody) and SW1389-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Example 4-3-9. FIG. 3 is a diagram showing changes in the percentage of activated helper T cells in the spleen upon administration of the antibody. hNS-mFa55 at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 30 mg/kg, SW1389-mFa55 at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, 500 mg /kg was administered via the tail vein. On the 6th day after administration, the spleen was collected, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis is the ratio of activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) to CD45 ⁺ cells. The average value of n=3 is shown. Figure 18 shows the antitumor effect of anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Example 5-4-5. It is a diagram. Antibodies were administered via the tail vein at 0.3 mg/kg, 1 mg/kg, and 3 mg/kg. Each point represents the average tumor volume of a group of n=5. Figure 19 shows the antitumor effect of anti-CTLA-4 antibody SW1612-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Example 5-4-5. It is a diagram. Antibodies were administered via the tail vein at 0.3 mg/kg, 1 mg/kg, and 3 mg/kg. Each point represents the average tumor volume of a group of n=5. Figure 20 shows the antitumor effect of anti-CTLA-4 antibody SW1615-mFa55 (switch antibody) in a mouse model transplanted with Hepa1-6/hGPC3 cell line, as described in Example 5-4-5. It is a diagram. Antibodies were administered via the tail vein at 0.3 mg/kg, 1 mg/kg, and 3 mg/kg. Each point represents the average tumor volume of a group of n=5. Figure 21 shows anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55 and SW1615-mFa55 (all FIG. 3 is a diagram showing changes in the percentage of effector Treg cells within a tumor upon administration of a switch antibody. SW1610-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, SW1612-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, SW1615-mFa55 at 50 mg/kg, 100 mg/ kg, 200 mg/kg, 400 mg/kg, and the negative control antibody KLH-mFa55 was administered at 400 mg/kg via the tail vein. On the 6th day after administration, tumor specimens were collected and FACS analysis was performed to evaluate the increase or decrease in effector Tregs. The vertical axis is the ratio of effector Tregs (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1 ⁺ ) to CD45 ⁺ cells. The average value of n=3 is shown. Figure 22 shows anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55 and SW1615-mFa55 (all This figure shows the change in the percentage of activated helper T cells in the spleen upon administration of the switch antibody. SW1610-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, SW1612-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, SW1615-mFa55 at 50 mg/kg, 100 mg/ kg, 200 mg/kg, 400 mg/kg, and the negative control antibody KLH-mFa55 was administered at 400 mg/kg via the tail vein. The spleen was collected on the 6th day after administration, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis is the ratio of activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) to CD45 ⁺ cells. The average value of n=3 is shown. FIG. 23 is a diagram showing a comparison of in vitro ADCC activities of antibodies having various modified constant regions with enhanced binding to FcγR, as described in Example 6-2. In the diagram, IgG1 is MDX10D1H-G1m/MDX10D1L-k0MT, GASDALIE is MDX10D1H-GASDALIE/MDX10D1L-k0MT, ART6 is MDX10D1H-Kn462/MDX10D1H-Hl445/MDX10D1L-k0MT, ART8 is MDX10D1H-K n461/MDX10D1H -Hl443/MDX10D1L-k0MT respectively. Here, IgG1 is an antibody with a control constant region, GASDALIE is an antibody with a constant region described in a prior document, and ART6 and ART8 are antibodies with modified constant regions prepared in Example 6-1. be. FIG. 24 is a diagram showing a comparison of in vitro ADCP activities of antibodies having various modified constant regions with enhanced binding to FcγR, as described in Example 6-3. In the diagram, IgG1 is MDX10D1H-G1m/MDX10D1L-k0MT, GASDIE is MDX10D1H-GASDIE/MDX10D1L-k0MT, ART6 is MDX10D1H-Kn462/MDX10D1H-Hl445/MDX10D1L-k0MT, ART8 is MDX10D1H-Kn4 61/MDX10D1H -Hl443/MDX10D1L-k0MT respectively. Here, IgG1 is an antibody with a control constant region, GASDIE is an antibody with a constant region described in the prior literature, and ART6 and ART8 are antibodies with modified constant regions prepared in Example 6-1. be. FIG. 25 shows the in vitro ADCC activity of anti-CTLA4 switch antibody SW1389-ART6 having a modified constant region with enhanced binding to FcγR, as described in Example 6-4. FIG. 26 shows the in vitro ADCC activity of anti-CTLA4 switch antibody SW1610-ART6 having a modified constant region with enhanced binding to FcγR, as described in Example 6-4. FIG. 27 shows the in vitro ADCC activity of anti-CTLA4 switch antibody SW1612-ART6 having a modified constant region with enhanced binding to FcγR, as described in Example 6-4. Figure 28 shows the neutralizing activity of anti-CTLA4 switch antibody SW1389 against CTLA4 (activity to release CTLA4 signals that suppress the activation of effector cells), as described in Example 6-5. FIG. Figure 29 shows the neutralizing activity of anti-CTLA4 switch antibody SW1610 against CTLA4 (activity to release CTLA4 signals that suppress the activation of effector cells), as described in Example 6-5. FIG. Figure 30 shows the neutralizing activity of anti-CTLA4 switch antibody SW1612 against CTLA4 (activity to release CTLA4 signals that suppress the activation of effector cells), as described in Example 6-5. FIG. Figure 31 shows the neutralizing activity of anti-CTLA4 switch antibody SW1615 against CTLA4 (activity to release CTLA4 signals that suppress the activation of effector cells), as described in Example 6-5. FIG. FIG. 32 is a diagram showing the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1389-ART5+ACT1 against CTLA4-positive regulatory T cells, as described in Example 6-6. FIG. 33 is a diagram showing the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1389-ART6+ACT1 against CTLA4-positive regulatory T cells, as described in Example 6-6. FIG. 34 is a diagram showing the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1610-ART5+ACT1 against CTLA4-positive regulatory T cells, as described in Example 6-6. Figure 35 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1610-ART6+ACT1 against CTLA4-positive regulatory T cells, as described in Example 6-6.

The techniques and procedures described or cited herein are generally well understood and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al. al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds. ., J.B. Lippincott Company, 1993) and are commonly used by those skilled in the art.

I. DEFINITIONS Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York , NY 1992) provides those skilled in the art with general guidance to many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

For purposes of interpreting this specification, the following definitions will apply and whenever applicable, terms used in the singular shall include the plural and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. If any of the definitions below conflict with any document incorporated herein by reference, the definitions below shall control.

An "acceptor human framework" for the purposes of this specification is a light chain variable domain (VL) framework or a heavy chain variable domain (VH ) is a framework, including the amino acid sequence of the framework. Acceptor human frameworks "derived from" human immunoglobulin frameworks or human consensus frameworks may contain the same amino acid sequences or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” (antibody-dependent cell-mediated cytotoxicity) is a method in which secreted immunoglobulins are released onto specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages). Fc receptors (FcRs) present in the cells, thereby allowing these cytotoxic effector cells to specifically bind to antigen-bearing target cells, and subsequently kill the target cells by cytotoxins. A form of cell damage that allows NK cells, the primary cells mediating ADCC, express only FcγRIII, and monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in US Pat. No. 5,500,362 or 5,821,337 or US Pat. No. 6,737,056 (Presta). Effector cells useful for such assays include PBMCs and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest may be assessed in vivo in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998). .

Examples of "cytotoxic activity" include the above-mentioned antibody-dependent cell-mediated cytotoxicity (ADCC) activity, the below-mentioned complement-dependent cytotoxicity (CDC) activity, and cytotoxic activity by T cells. CDC activity means cytotoxic activity by the complement system. On the other hand, ADCC activity refers to an activity in which an antibody binds to an antigen present on the cell surface of a target cell, and the effector cell further binds to the antibody, thereby causing the effector cell to cause damage to the target cell. Whether the antibody of interest has ADCC activity or CDC activity can be determined by a known method (for example, Current Protocols in Immunology, Chapter 7. Immunologic studies in humans, edited by Coligan et al. (1993)). etc).

"Neutralizing activity" refers to the activity of inhibiting a biological activity when an antibody binds to a molecule involved in that biological activity. In some embodiments, biological activity results from binding of a ligand to a receptor. In certain embodiments, the antibody inhibits binding of the ligand or receptor by binding to the ligand or receptor. Antibodies with such neutralizing activity are called neutralizing antibodies. The neutralizing activity of a given analyte can be determined by comparing the biological activity in the presence of the ligand between conditions in the presence or absence of the analyte.

The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells).

The term "binding activity" refers to the noncovalent interaction between one or more binding sites of a molecule (e.g., an antibody) and the molecule's binding partner (e.g., an antigen). Refers to the total strength. Here, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (eg, an antibody and an antigen). For example, avidity refers to the inherent binding affinity (“affinity”) when the members of a binding pair reflect a 1:1 interaction with monovalents. When members of a binding pair are capable of both monovalent and multivalent binding, avidity is the sum of these avidities. The binding activity of molecule X toward its partner Y can generally be expressed by a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand." Binding activity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding activity are discussed below.

An "affinity matured" antibody has one or more modifications in one or more hypervariable regions (HVR) that result in improved affinity of the antibody for antigen compared to a parent antibody without the modifications. Refers to antibodies that are associated with

The term "anti-CTLA-4 antibody" or "antibody that binds CTLA-4" refers to an antibody that is capable of binding CTLA-4 with sufficient affinity such that the antibody targets CTLA-4. refers to antibodies that are useful as diagnostic and/or therapeutic agents when In one embodiment, the extent of binding of an anti-CTLA-4 antibody to an unrelated non-CTLA-4 protein is determined by the binding of the antibody to CTLA-4 as measured (e.g., by radioimmunoassay (RIA)). less than about 10% of In certain embodiments, the antibody that binds CTLA-4 is ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 ^-8 M or less). , eg 10 ^-8 M to 10 ^-13 M, eg 10 ^-9 M to 10 ^-13 M). In certain embodiments, the anti-CTLA-4 antibody binds to an epitope of CTLA-4 that is conserved among CTLA-4 from different species.

The term "antibody" is used herein in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., It encompasses a variety of antibody structures, including bispecific antibodies) and antibody fragments.

"Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; diabodies; linear antibodies; single chain antibody molecules (e.g., scFv ); and multispecific antibodies formed from antibody fragments.

An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that blocks the reference antibody's binding to its own antigen by, for example, 50% or more in a competitive assay, and/or In this case, the above-mentioned antibody binds to its own antigen by, for example, 50% or more. An exemplary competition assay is provided herein.

"Autoimmune disease" refers to a non-malignant disease or disorder that arises from and is directed against an individual's own tissues. As used herein, autoimmune disease specifically excludes malignant or cancerous diseases or conditions, in particular B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic exclude chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia. Examples of autoimmune diseases or disorders include, but are not limited to: inflammatory reactions such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); Scleroderma and sclerosis; reactions associated with inflammatory bowel diseases (e.g. Crohn's disease and ulcerative colitis); respiratory distress syndromes (including adult respiratory distress syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions with T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiencies; joints rheumatism; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); diabetes (e.g., type I diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud syndrome; by immune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile-onset diabetes; and by cytokines and T lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis. Immune reactions associated with mediated acute and delayed hypersensitivity; pernicious anemia (Addison's disease); diseases with extravasation of leukocytes; central nervous system (CNS) inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; stiff man syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA Nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include breast cancer and liver cancer.

The term "complement-dependent cytotoxicity" or "CDC" refers to a cell death process in which the Fc effector domain of an antibody bound to a target activates a series of enzymatic reactions that result in the formation of holes in the membrane of the target cell. means a mechanism for inducing Typically, antigen-antibody complexes formed on target cells bind and activate complement component C1q, which in turn activates the complement cascade, resulting in the death of the target cell. bring about. Activation of complement may also result in the deposition of complement components on the surface of target cells, which promotes ADCC by binding to complement receptors (e.g. CR3) on leukocytes. .

"Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; benzodopa, carbocon aziridines, such as , meturedopa, and uredopa; ethyleneimines and methylolmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; acetogenins, especially buratacine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analog topotecan (HYCAMTIN®)), CPT-11 (including irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its adzeresin, calzelesin, and vizeresin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); elouterobin; pancratistatin; Cutiin; spongistatin; chlorambucil, chlornafadine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbikin, fenesterine, prednimustine, trofosfamide, uracil mustard, etc. Nitrogen mustards, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics {e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; zinemycins, including zinemycin A; esperamycins; and neocarzinostatin chromophore and related chromoproteins enediyne antibiotics chromophore, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calubicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin , detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl for liposome injection (DOXIL®) ), liposomal doxorubicin TCL D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, Antibiotics such as mycophenolic acid, nogaramycin, olibomycin, pepromycin, porphyromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; methotrexate, Antimetabolites such as gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone, and 5-fluorouracil (5-FU); denopterin, methotrexate, pteropterin , folic acid analogs such as trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamipurine, thioguanine; , pyrimidine analogs; androgens, such as carsterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; anti-adrenals, such as aminoglutethimide, mitotane, trilostane; folic acid supplementation, such as folinic acid. Substances; acegratone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabcil; bisanthrene; edatrexate; defofamine; demecoltine; diaziquon; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamole; nitraerine; pentostatin; phenamet; pirarubicin; rosoxantrone; 2-ethylhydrazide; procarbazine; PSK ® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitractol; pipobroman; gacytosine; arabinoside (“Ara -C”); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE®), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercapto purines; methotrexate; platinum agents, such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®); ), FILDESIN®) and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); Trademark) or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), bisphosphonates, such as tiludronate (SKELID®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, especially unusual those that inhibit the expression of genes in signal transduction pathways associated with cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); THERATOPE® Vaccines, such as vaccines and gene therapy vaccines (e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine); topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g. ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g. celecoxib or etoricoxib), proteosome inhibition agents (e.g., PS341); Bcl-2 inhibitors, such as bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); sorafenib, ABT510; oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definitions below); tyrosine kinase inhibitors (see definitions below); serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE®); lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as cyclophosphamide. , CHOP, which stands for combination therapy of doxorubicin, vincristine, and prednisolone; and FOLFOX, which stands for treatment regimen with oxaliplatin (ELOXANTIN™) in combination with 5-FU and leucovorin.

The term "chimeric" antibody refers to antibodies in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remaining portion of the heavy and/or light chain is derived from a different source or species. Refers to antibodies.

The "class" of an antibody refers to the type of constant domain or constant region present in the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes death or destruction of cells. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and radioactive isotopes of Lu); chemotherapeutic agents or chemotherapeutic agents (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalators); growth inhibitors; enzymes such as nucleolytic enzymes and fragments thereof; antibiotics; and the various chemotherapeutic agents disclosed above.

"Effector cells" refer to leukocytes that express one or more FcRs and exert effector functions. In certain embodiments, the cell expresses at least FcγRIII and exerts ADCC effector function. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood. In certain embodiments, effector cells can be human effector cells.

"Effector function" refers to the biological activity attributable to the Fc region of an antibody that differs depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (CDC); cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation .

The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody targeting that antigen and includes specific amino acids that are in direct contact with the antibody. Epitopic determinants can include chemically active surface molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and have specific three-dimensional structural characteristics and/or specific charge characteristics. I can do it. Generally, antibodies specific for a particular target antigen preferentially recognize epitopes on that target antigen in a complex mixture of proteins and/or macromolecules.

"Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR is one that binds IgG antibodies (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. including, containing. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitory receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). thing). FcR has been described, for example, by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin Reviewed in Med 126: 330-341 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

The term "Fc receptor" or "FcR" also refers to the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) as well as the neonatal receptor FcRn, which is responsible for regulating immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (eg, Ghetie and Ward., Immunol. Today 18(12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7): 637- 640 (1997); Hinton et al., J. Biol. Chem. 279(8): 6213-6216 (2004); WO2004/92219 (Hinton et al.).

The binding to human FcRn in vivo and the serum half-life of human FcRn high affinity binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or when polypeptides with mutant Fc regions are can be measured in the treated primate. WO2000/42072 (Presta) describes antibody variants with improved or decreased binding to FcR. See also, eg, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001).

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in Fc or constant regions is as per Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, It follows the EU numbering system (also known as the EU index) as described in MD 1991.

The term "Fc region-containing antibody" refers to an antibody that includes an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine of the Fc region (residues 446-447) can be used, for example, during antibody purification or in the nucleic acid encoding the antibody. can be removed by recombinant manipulation. Therefore, a composition comprising an antibody having an Fc region according to the present invention can be an antibody with G446-K447, an antibody with G446 and no K447, an antibody with G446-K447 completely removed, or an antibody of the above three types. may include a mixture of.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is responsible for binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of naturally occurring antibodies are typically similar, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). structure (see, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). One VH or VL domain may be sufficient to confer antigen binding specificity. Additionally, antibodies that bind to a particular antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, eg, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

"Framework" or "FR" refers to variable domain residues other than the hypervariable region (HVR) residues. The variable domain FR usually consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and have a structure substantially similar to a naturally occurring antibody structure, or as defined herein. An antibody that has a heavy chain that includes an Fc region.

A "functional Fc region" comprises the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (eg, B cell receptors (BCR)), and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using, e.g., various assays disclosed within the definitions herein. can be done.

A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cells or derived from a non-human source using a human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues.

A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selected group of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, a subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III by Kabat et al., supra.

A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVR and from human FR. In certain embodiments, the humanized antibody comprises substantially all of at least one, typically two, variable domains in which all or substantially all HVRs (e.g., CDRs) are non-transparent. Corresponds to that of a human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to a region that is hypervariable in sequence ("complementarity determining region" or "CDR") and/or structurally defined. Refers to each region of an antibody's variable domain that forms loops (“hypervariable loops”) and/or contains antigen-contacting residues (“antigen-contacting”). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include:
(a) At amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) resulting hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));
(b) At amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) resulting CDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) At amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3). antigen contact that occurs (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
(d) HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49 Combinations of (a), (b), and/or (c), including -65 (H2), 93-102 (H3), and 94-102 (H3).
Unless otherwise indicated, HVR residues and other residues in variable domains (eg, FR residues) are numbered herein according to Kabat et al., supra.

An "immunoconjugate" is an antibody that is conjugated to one or more heterologous molecules (heterologous molecules include, but are not limited to, cytotoxic agents).

An "isolated" antibody is one that has been separated from the components of its native environment. In some embodiments, the antibody is purified, e.g., by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). Measured and purified to greater than 95% or 99% purity. For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its native environment. Isolated nucleic acid includes a nucleic acid molecule that is contained within a cell that normally contains the nucleic acid molecule, but that is located extrachromosomally or on a chromosome different from its natural chromosomal location. exists in position.

"Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, carried on one vector or on separate vectors. and nucleic acid molecules that are present in one or more locations in a host cell.

The term "vector" as used herein refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of the host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operably linked. Such vectors are also referred to herein as "expression vectors."

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell (including the progeny of such a cell) that has been introduced with a foreign nucleic acid. . Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Also included herein are mutant progeny that have the same function or biological activity for which the original transformed cell was screened or selected.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are free from any mutant antibodies that may arise (e.g., mutant antibodies that contain naturally occurring mutations, or that arise during the manufacture of monoclonal antibody preparations. Such mutants usually have some are identical and/or bind to the same epitope, except in the amount present). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, and without limitation, monoclonal antibodies used in accordance with the present invention may be produced using hybridoma technology, recombinant DNA technology, phage display technology, transgenic animals containing all or part of human immunoglobulin loci. Such and other exemplary methods for making monoclonal antibodies are described herein.

"Naked antibody" refers to an antibody that is not conjugated to a foreign moiety (eg, a cytotoxic moiety) or a radioactive label. Naked antibodies may be present in pharmaceutical formulations.

"Natural antibody" refers to immunoglobulin molecules with a variety of naturally occurring structures. For example, naturally occurring IgG antibodies are approximately 150,000 Dalton heterotetrameric glycoproteins composed of two identical light chains and two identical heavy chains that are disulfide bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Antibody light chains may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

A "native sequence Fc region" includes an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include native-sequence human IgG1 Fc regions (non-A and A allotypes); native-sequence human IgG2 Fc regions; native-sequence human IgG3 Fc regions; and native-sequence human IgG4 Fc regions; including those variants that exist in

A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the variant Fc region contains at least one amino acid substitution in the native sequence Fc region or in the Fc region of the parent polypeptide as compared to the native sequence Fc region or the Fc region of the parent polypeptide, e.g. It has about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions. The variant Fc regions herein preferably have at least about 80% homology to the native sequence Fc region and/or the Fc region of the parent polypeptide, more preferably at least about 90% homology thereto, and most preferably have at least about 95% homology with them.

"Percent (%) Amino Acid Sequence Identity" to a reference polypeptide sequence refers to the sequence identity after aligning the sequences to obtain the maximum percent sequence identity and introducing gaps, if necessary, and after any conservative substitutions have been made. It is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, when not considered part of the identity. Alignments for the purpose of determining percent amino acid sequence identity can be performed using a variety of methods within the skill in the art, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (stock This can be accomplished using publicly available computer software, such as the company Genetics. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared.

The ALIGN-2 sequence comparison computer program is the copyrighted work of Genentech, Inc., the source code of which has been submitted with user documentation to the US Copyright Office, Washington DC, 20559, under US Copyright Registration No. TXU510087. Registered. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.
In situations where ALIGN-2 is used for amino acid sequence comparisons, the percent amino acid sequence identity of a given amino acid sequence A to or with a given amino acid sequence B (or A given amino acid sequence A, which has or contains a certain % amino acid sequence identity to, with, or to B) is calculated as follows: of the fraction X/Y. Hundredfold. where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignments of A and B, and Y is the total number of amino acid residues in B. . It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Dew. Unless otherwise specified, all % amino acid sequence identity values used herein are those obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

The term "pharmaceutical preparation" means a preparation in such a form that the biological activity of the active ingredient contained therein can be exerted, and to an extent unacceptable to the subject to whom the preparation is administered. A preparation that does not contain additional toxic elements.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and , including rodents (e.g. mice and rats). In certain embodiments, the individual or subject is a human.

"Pharmaceutically acceptable carrier" refers to an ingredient other than the active ingredient in a pharmaceutical formulation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to that amount, at doses and for periods of time necessary, that is effective to achieve the desired therapeutic or prophylactic result. .

The term "package insert" refers to the information normally included in the commercial packaging of therapeutic products and containing information about indications, dosage, dosage, methods of administration, concomitant therapy, contraindications, and/or warnings regarding the use of such therapeutic products. Used to refer to instruction manuals.

The term "CTLA-4" as used herein, unless otherwise indicated, is from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). refers to any natural form of CTLA-4. The term encompasses "full length" unprocessed CTLA-4 as well as any form of CTLA-4 that results from processing in the cell. The term also encompasses naturally occurring variants of CTLA-4, such as splice variants and allelic variants. Exemplary human CTLA-4 amino acid sequence is SEQ ID NO: 214, mouse CTLA-4 amino acid sequence is SEQ ID NO: 247, monkey CTLA-4 amino acid sequence is SEQ ID NO: 248, and human CTLA-4 amino acid sequence is SEQ ID NO: 247. The amino acid sequence of the extracellular domain is shown in SEQ ID NO: 28. In this specification, CTLA-4 may also be referred to as CTLA4.

The term "regulatory T (Treg) cells" is a subpopulation of T cells that regulates the immune system, maintains tolerance to self-antigens, and suppresses autoimmune diseases. These cells generally suppress or downregulate effector T cell induction and proliferation. The best understood Treg cells are those that express CD4, CD25, and Foxp3 (CD4 ⁺ CD25 ⁺ Treg cells). These Tregs are different from helper T cells. Several different methods are used to identify and monitor Treg cells. As defined by CD4 and CD25 expression (CD4 ⁺ CD25 ⁺ cells), Treg cells constitute approximately 5-10% of the mature CD4 ⁺ T cell subpopulation in mice and humans, whereas approximately 1-2% of Tregs Can be measured in whole blood. Foxp3 expression may also be measured (CD4 ⁺ CD25 ⁺ Foxp3 ⁺ cells). The absence or low level expression of CD127 may also be used in combination with the presence of CD4 and CD25 as another marker. Additionally, Treg cells also express high levels of CTLA-4 and GITR. Tregs can also be identified by the method described in the Examples below.

As used herein, the term "substantially similar," "substantially equal," or "substantially the same" refers to the relationship between two numbers (e.g., with respect to an antibody of the invention and a reference/comparison). (with respect to antibodies for use), it is clear to those skilled in the art that the difference between those two values has little or no biological and/or Or, it is sufficiently high that it is considered to have no statistical significance.

As used herein, "therapy" (and its grammatical derivatives, e.g., "treat," "treating," etc.) refers to clinical treatment intended to alter the natural history of the individual being treated. It refers to an intervention, which can be carried out both for prevention and during the course of a clinical condition. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological effects of the disease, preventing metastasis, including reduction in the rate of progression, reversal or mitigation of disease status, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay the onset of a disease or slow the progression of a disease.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" as used herein are not mutually exclusive.

The term "tumor tissue" means tissue that contains at least one tumor cell. Tumor tissue usually consists of a group of tumor cells (parenchyma) that forms the main body of the tumor, and connective tissue and blood vessels (stroma) that exist between these cells and support the tumor. In some cases, the distinction between the two is clear, while in others, the two are mixed together. Immune cells may also be infiltrated within the tumor tissue. On the other hand, "non-tumor tissue" means tissue other than tumor tissue in vivo. Healthy/normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

II. Compositions and Methods In one aspect, the invention is based in part on anti-CTLA-4 antibodies and uses thereof. In certain embodiments, antibodies that bind CTLA-4 are provided. The antibodies of the invention are useful, for example, for the diagnosis or treatment of cancer.

A. Exemplary Anti-CTLA-4 Antibodies In one aspect, the invention provides isolated antibodies that bind CTLA-4. In certain embodiments, anti-CTLA-4 antibodies of the invention have CTLA-4 binding activity that is dependent on the concentration of adenosine-containing compounds. In some embodiments, the binding activity to CTLA-4 is greater in the presence of an adenosine-containing compound than in the absence of an adenosine-containing compound. Alternatively, in another embodiment, the binding activity to CTLA-4 is higher in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in binding activity to CTLA-4 is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more. More than 300 times, 500 times or more, 1 × 10 ³ times or more, 2 × 10 3 times or more, 3 × 10 ³ times or more, 5 × 10 ³ times ^or more, 1 × 10 ⁴ times or more, 2 × 10 ⁴ times or more, 3×10 ⁴ times or more, 5×10 ⁴ times or more, or 1×10 ⁵ times or more.

In some embodiments, the binding activity of an anti-CTLA-4 antibody can be expressed as a KD (Dissociation constant) value. In a further embodiment, the KD value of the anti-CTLA-4 antibody is lower in the presence of an adenosine-containing compound compared to the absence of an adenosine-containing compound. Alternatively, in another embodiment, the KD value of the anti-CTLA-4 antibody is lower in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in KD values of anti-CTLA-4 antibodies is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more. 3x or more, 300x or more, 500x or more, 1x10 ^3x or more, 2x10 3x or more, 3x10 ^{3x or} more, 5x10 ^{3x or} more, 1x10 ^4x or more, 2x10 ⁴ 3×10 ⁴ times or more, 5×10 ⁴ times or more, or 1×10 ⁵ times or more. The KD value of anti-CTLA-4 antibody in the presence of an adenosine-containing compound or in the presence of a high concentration of an adenosine-containing compound is, for example, 9 × 10 ^-7 M or less, 8 × 10 ^-7 M or less, 7 × 10 ^-7 M or less, 6×10 ^-7 M or less, 5×10 ^-7 M or less, 4×10 ^-7 M or less, 3×10 ^-7 M or less, 2×10 ^-7 M or less, 1×10 ^-7 M or less , 9×10 ^-8 M or less, 8×10 ^-8 M or less, 7×10 ^-8 M or less, 6×10 ^-8 M or less, 5×10 ^-8 M or less, 4×10 ^-8 M or less, 3 ×10 ^-8 M or less, 2 × 10 ^-8 M or less, 1 × 10 ^-8 M or less, 9 × 10 ^-9 M or less, 8 × 10 -9 M or less, 7 × ^{10 -9} M or less, 6 × 10 ^-9 M or less, 5×10 ^-9 M or less, 4×10 ^-9 M or less, 3×10 ^-9 M or less, 2×10 ^-9 M or less, 1×10 ^-9 M or less, 9×10 ^-10 M or less, 8×10 ^-10 M or less, 7×10 ^-10 M or less, 6×10 ^-10 M or less, 5×10 -10 M or less, 4×10 ^{-10 M} or less, 3×10 ^-10 M or less , 2×10 ⁻¹⁰ M or less, or 1×10 ⁻¹⁰ M or less. The KD value of anti-CTLA-4 antibody in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound is, for example, 1 × 10 ^-8 M or more, 2 × 10 ^-8 M or more, 3 × 10 ^{- 8} M or more, 4 × 10 ^-8 M or more, 5 × 10 ^-8 M or more, 6 × 10 ^-8 M or more, 7 × 10 ^-8 M or more, 8 × 10 ^-8 M or more, 9 × 10 ^-8 M 1 x 10 ^-7 M or more, 2 x 10 ^-7 M or more, 3 x 10 ^-7 M or more, 4 x 10 ^-7 M or more, 5 x 10 ^-7 M or more, 6 x 10 ^-7 M or more, 7×10 ^-7 M or more, 8×10 ^-7 M or more, 9×10 ^-7 M or more, 1×10 ^-6 M or more, 2×10 -6 M or more, 3× ^{10 -6 M} or more, 4× 10 ^-6 M or more, 5 × 10 ^-6 M or more, 6 × 10 ^-6 M or more, 7 × 10 ^-6 M or more, 8 × 10 ^-6 M or more, or 9 × 10 ^-6 M or more can.

In another embodiment, the binding activity of the anti-CTLA-4 antibody may be expressed by a kd (Dissociation rate constant) value instead of a KD value.

In another embodiment, the binding activity of an anti-CTLA-4 antibody may be expressed as the amount of CTLA-4 bound per unit amount of antibody. For example, in a surface plasmon resonance assay, the amount of bound antibody immobilized on a sensor chip and the amount of antigen bound thereto are each measured as a resonance unit (RU). The value obtained by dividing the amount of antigen bound by the amount of antibody bound can be defined as the amount of antigen bound per unit amount of antibody. Specific methods for measuring and calculating such binding amount are described in Examples below. In some embodiments, the amount of CTLA-4 bound is greater in the presence of an adenosine-containing compound than in the absence of an adenosine-containing compound. Alternatively, in another embodiment, the amount of CTLA-4 bound is greater in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in the amount of CTLA-4 bound is, for example, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more , 300x or more, 500x or more, 1x10 ^3x or more, 2x10 3x or more, 3x10 ^{3x or} more, 5x10 ^3x or more, 1x10 ^4x or more, 2x10 ^4x or more , 3×10 ⁴ times or more, 5×10 ⁴ times or more, or 1×10 ⁵ times or more. The binding amount of CTLA-4 in the presence of an adenosine-containing compound or a high concentration of an adenosine-containing compound is, for example, 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, It can be 0.08 or more, 0.09 or more, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, or 1 or more. The binding amount of CTLA-4 in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound is, for example, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less. , 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, 0.003 or less, 0.002 or less, or 0.001 or less Something can happen.

In some embodiments, the KD value, kd value, binding amount value, etc. expressed herein are measured or calculated by performing a surface plasmon resonance assay at 25°C or 37°C (e.g., See Example 3 herein).

Any concentration of the adenosine-containing compound can be selected as long as a difference in the binding activity of the anti-CTLA-4 antibody is detected. In certain embodiments, high concentrations include, for example, 1 nM or higher, 3 nM or higher, 10 nM or higher, 30 nM or higher, 100 nM or higher. , 300 nM or higher, 1 μM or higher, 3 μM or higher, 10 μM or higher, 30 μM or higher, 100 μM or higher, 300 μM or higher, 1 mM or higher, 3 mM or higher, 10 mM or higher, 30 mM or higher, 100 mM or higher, 300 mM or Higher concentrations may be mentioned, such as 1 M or higher. Alternatively, the concentration can be high enough that each anti-CTLA-4 antibody exhibits maximum binding activity. In one embodiment, the high concentration can be selected to be 1 μM, 10 μM, 100 μM, 1 mM, or a sufficient amount such that the respective anti-CTLA-4 antibody exhibits maximum binding activity. In certain embodiments, low concentrations include, for example, 1 mM or lower, 300 μM or lower, 100 μM or lower, 30 μM or lower, 10 μM or lower. , 3 μM or lower, 1 μM or lower, 300 nM or lower, 100 nM or lower, 30 nM or lower, 10 nM or lower, 3 nM or lower, 1 nM or lower, 300 pM or lower, 100 pM or lower, 30 pM or lower, 10 pM or lower, 3 pM or lower concentrations, such as 1 pM or lower. Alternatively, the concentration at which each anti-CTLA-4 antibody exhibits the minimum binding activity can be set as the low concentration. A substantially zero concentration (in the absence of an adenosine-containing compound) can also be selected as one embodiment of low concentration. In one embodiment, the low concentration is selected to be 1 mM, 100 μM, 10 μM, 1 μM, the concentration at which the respective anti-CTLA-4 antibody exhibits minimal binding activity, or in the absence of an adenosine compound. be able to. In another embodiment, the ratio of high concentration to low concentration is, for example, 3 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times or more, 1×10 ³ times or more, 3x10 ^3x or more, 1x10 ^4x or more, 3x10 ^4x or more, 1x10 ^5x or more, 3x10 ^5x or more, 1x10 ^6x or more, 3x10 ^6x or more, 1x10 ^7x or more, 3x10 ^7x or more, 1x10 ^8x or more, 3x10 ^8x or more, 1x10 ^9x or more, 3x10 ^9x or more, 1x10 ^10x or more, 3x10 ^10x or more, 1x10 ^11x or more, 3 You can choose a value of ×10 ¹¹ times or more, 1 × 10 ¹² times or more.

In another embodiment, the anti-CTLA-4 antibody of the invention also has binding activity for adenosine-containing compounds. Using the method described above, the amount of adenosine-containing compound bound per unit amount of antibody can be calculated, and this can be taken as the binding activity of the anti-CTLA-4 antibody to the adenosine-containing compound. Specific methods for measuring and calculating such binding amount are described in Examples below. The binding amount of the adenosine-containing compound per unit antibody amount of the anti-CTLA-4 antibody of the present invention is, for example, 0.0001 or more, 0.0002 or more, 0.0003 or more, 0.0004 or more, 0.0005 or more, 0.0006 or more, 0.0007 or more, 0.0008 or more, 0.0009. 0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, or 0.01 or more.

In another embodiment, an anti-CTLA-4 antibody of the invention forms a ternary complex with an adenosine-containing compound and CTLA-4. In one embodiment, the anti-CTLA-4 antibody binds the adenosine-containing compound through heavy chain CDR1, CDR2, CDR3. In one embodiment, the anti-CTLA-4 antibody has a binding motif for an adenosine-containing compound. Binding motifs for adenosine-containing compounds are present, for example, at positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c according to Kabat numbering. It may be composed of at least one amino acid. In a further embodiment, the anti-CTLA-4 antibody is located at positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, 100c, e.g., according to Kabat numbering. binds to an adenosine-containing compound via at least one amino acid selected from the group consisting of: In certain embodiments, the anti-CTLA-4 antibody has the following Kabat numbering: Thr at position 33, Ser at position 52, Ser at position 52a, Arg at position 53, Tyr at position 56, Tyr at position 58, and position 95. Tyr at position 96, Gly at position 96, Met at position 100a, Leu at position 100b, and Trp at position 100c. CTLA-4 may further bind to the complex formed by binding the anti-CTLA-4 antibody and the adenosine-containing compound. Furthermore, the adenosine-containing compound may be present at the interface where the anti-CTLA-4 antibody and CTLA-4 interact and bind to both of them. Formation of a ternary complex by the anti-CTLA-4 antibody with an adenosine-containing compound and CTLA-4 can be confirmed, for example, by techniques such as crystal structure analysis described below (see Examples).

In another embodiment, the anti-CTLA-4 antibody of the present invention comprises amino acids 3rd (Met), 33rd amino acids (Glu), and 35th amino acids of human CTLA-4 (extracellular domain, SEQ ID NO: 28). (Arg), 53rd amino acid (Thr), 97th amino acid (Glu), 99th amino acid (Met), 100th amino acid (Tyr), 101st amino acid (Pro), 102nd amino acid (Pro ), the 103rd amino acid (Pro), the 104th amino acid (Tyr), the 105th amino acid (Tyr), and the 106th amino acid (Leu). These amino acids may constitute the epitope of the anti-CTLA-4 antibody of the invention. In another embodiment, the anti-CTLA-4 antibody of the present invention binds to a region from amino acid 97 (Glu) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). . In another embodiment, the anti-CTLA-4 antibody of the present invention binds to a region from amino acid 99 (Met) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). .

In another embodiment, the anti-CTLA-4 antibodies of the invention are characterized in that ABAM004 (VH, SEQ ID NO: 10; VL, SEQ ID NO: 11; HVR-H1, SEQ ID NO: 100; HVR- H2, SEQ ID NO: 101; HVR-H3, SEQ ID NO: 102; HVR-L1, SEQ ID NO: 113; HVR-L2, SEQ ID NO: 114; HVR-L3, SEQ ID NO: 115). In another embodiment, the anti-CTLA-4 antibodies of the invention bind to the same epitope as ABAM004. When anti-CTLA-4 antibodies are present in excess, reduce the binding of ABAM004 to CTLA-4 by, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more. , 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or can be reduced by 95% or more . An exemplary competition assay is provided herein.

In another embodiment, the anti-CTLA-4 antibodies of the invention exhibit cytotoxic activity against CTLA-4 expressing cells. If CTLA-4 is expressed on the surface of a target cell and an anti-CTLA-4 antibody binds thereto, the cell can be damaged. Injury to cells is caused by effector cells bound to antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). It may be caused by complement binding to the antibody, such as complement-dependent cytotoxicity (CDC). Alternatively, it may be caused by a cytotoxic agent (eg, a radioisotope, a chemotherapeutic agent, etc.) bound to an antibody, such as an immunoconjugate. The cell injury here may include an effect of inducing cell death, an effect of suppressing cell proliferation, an effect of impairing cell function, and the like. When anti-CTLA-4 antibodies are present in a sufficient amount, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more , causing damage to 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of CTLA-4 expressing cells. I can do it. Such measurements of cytotoxic activity can be made in comparison to measurements in the absence of the antibody or in the presence of a negative control antibody. Exemplary cytotoxicity assays are provided herein.

In another embodiment, the anti-CTLA-4 antibodies of the invention exhibit neutralizing activity against CTLA-4. CTLA-4 is known to function by interacting with its ligands CD80 (B7-1) or CD86 (B7-2). In certain embodiments, the anti-CTLA-4 antibody inhibits the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2). When anti-CTLA-4 antibodies are present in sufficient amounts, the interaction between CTLA-4 and CD80 (B7-1) or CD86 (B7-2) can be reduced by, for example, 10% or more, 15% or more, 20% or more, 25% or more. % or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more , 90% or more, or 95% or more. Such inhibitory activity measurements can be made in comparison to measurements in the absence of the antibody or in the presence of a negative control antibody. Specific methods of measuring neutralizing activity are provided herein.

In another embodiment, the anti-CTLA-4 antibodies of the invention bind CTLA-4 from multiple animal species. Exemplary animal species can include mammals, such as humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, horses, sheep, camels, dogs, cats, and the like. In certain embodiments, anti-CTLA-4 antibodies bind CTLA-4 from humans and non-humans (eg, monkeys, mice, rats, etc.). The amino acid sequence of human CTLA-4 is shown in SEQ ID NO: 214, the amino acid sequence of monkey CTLA-4 is shown in SEQ ID NO: 247, and the amino acid sequence of mouse CTLA-4 is shown in SEQ ID NO: 248. The amino acid sequence of CTLA-4 derived from other animal species can also be appropriately determined by methods known to those skilled in the art.

In certain embodiments, the adenosine-containing compounds of the present invention include, for example, adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and cyclic adenosine monophosphate. (cAMP), deoxyadenosine (dADO), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), adenosine gamma thiotriphosphate (ATPγS), etc. I can do it.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; and (c) the amino acid sequence of SEQ ID NO: 225. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; (c) amino acid sequence of SEQ ID NO: 225. HVR-H3 containing the sequence.

In another aspect, the invention provides (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 228. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; (c) amino acid sequence of SEQ ID NO: 228. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 224. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227, and (c) comprising the amino acid sequence of SEQ ID NO: 228. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; (c) SEQ ID NO: 225 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 226; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 227; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 228.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; and (c) the amino acid sequence of SEQ ID NO: 102. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; (c) amino acid sequence of SEQ ID NO: 102. HVR-H3 containing the sequence.

In another aspect, the invention provides: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; (c) the amino acid sequence of SEQ ID NO: 115. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 101. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) comprising the amino acid sequence of SEQ ID NO: 115. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; (c) SEQ ID NO: 102 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 113; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 114; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 115.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; and (c) the amino acid sequence of SEQ ID NO: 102. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; (c) amino acid sequence of SEQ ID NO: 102. HVR-H3 containing the sequence.

In another aspect, the invention provides: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; (c) amino acid sequence of SEQ ID NO: 115. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 104. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) comprising the amino acid sequence of SEQ ID NO: 115. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; (c) SEQ ID NO: 102 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 116; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 115.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; and (c) the amino acid sequence of SEQ ID NO: 102. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; (c) amino acid sequence of SEQ ID NO: 102. HVR-H3 containing the sequence.

In another aspect, the invention provides (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 133. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; (c) amino acid sequence of SEQ ID NO: 133. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 106. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) comprising the amino acid sequence of SEQ ID NO: 133. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; (c) SEQ ID NO: 102 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 133.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; and (c) the amino acid sequence of SEQ ID NO: 102. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; (c) amino acid sequence of SEQ ID NO: 102. HVR-H3 containing the sequence.

In another aspect, the invention provides: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 153. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; (c) amino acid sequence of SEQ ID NO: 153. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 108. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (c) comprising the amino acid sequence of SEQ ID NO: 153. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; (c) SEQ ID NO: 102 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 121; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 123; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 153.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) the amino acid sequence of SEQ ID NO: 102. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; (c) amino acid sequence of SEQ ID NO: 102. HVR-H3 containing the sequence.

In another aspect, the invention provides (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 133. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; (c) amino acid sequence of SEQ ID NO: 133. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 110. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) comprising the amino acid sequence of SEQ ID NO: 133. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; (c) SEQ ID NO: 102 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 133.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) the amino acid sequence of SEQ ID NO: 102. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; (c) the amino acid sequence of SEQ ID NO: 102 HVR-H3 containing the sequence.

In another aspect, the invention provides (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 133. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; (c) amino acid sequence of SEQ ID NO: 133. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 112. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) comprising the amino acid sequence of SEQ ID NO: 133. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; (c) SEQ ID NO: 102 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 133.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) the amino acid sequence of SEQ ID NO: 152. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) amino acid sequence of SEQ ID NO: 152. HVR-H3 containing the sequence.

In another aspect, the invention provides (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 133. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; (c) amino acid sequence of SEQ ID NO: 133. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 111. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) comprising the amino acid sequence of SEQ ID NO: 133. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) SEQ ID NO: 152 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 133.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) the amino acid sequence of SEQ ID NO: 102. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; (c) the amino acid sequence of SEQ ID NO: 102 HVR-H3 containing the sequence.

In another aspect, the invention provides: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 133. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; (c) amino acid sequence of SEQ ID NO: 133. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 112. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) comprising the amino acid sequence of SEQ ID NO: 133. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; (c) SEQ ID NO: 102 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 133.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) the amino acid sequence of SEQ ID NO: 152. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) amino acid sequence of SEQ ID NO: 152. HVR-H3 containing the sequence.

In another aspect, the invention provides: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 133. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; (c) amino acid sequence of SEQ ID NO: 133. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 111. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) comprising the amino acid sequence of SEQ ID NO: 133. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) SEQ ID NO: 152 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 133.

In one aspect, the invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; and (c) the amino acid sequence of SEQ ID NO: 102. Antibodies are provided that include at least one, at least two, or all three VH HVR sequences selected from HVR-H3 containing sequences. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; (c) amino acid sequence of SEQ ID NO: 102. HVR-H3 containing the sequence.

In another aspect, the invention provides (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 133. Antibodies are provided that include at least one, at least two, or all three VL HVR sequences selected from the amino acid sequences HVR-L3. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; (c) amino acid sequence of SEQ ID NO: 133. HVR-L3 containing the sequence.

In another aspect, the antibodies of the invention have (a) a VH domain, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 109. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (b) VL A domain comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) comprising the amino acid sequence of SEQ ID NO: 133. and a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from HVR-L3.

In another aspect, the present invention provides: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; (c) SEQ ID NO: 102 (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) SEQ ID NO: HVR-L3 comprising an amino acid sequence selected from 133.

In certain embodiments, any one or more amino acids of the anti-CTLA-4 antibodies described above are substituted at the following HVR positions:
-In HVR-H1 (SEQ ID NO: 223): Position 2
- In HVR-H2 (SEQ ID NO: 224): positions 4, 5, 7, 13, and 16
-In HVR-H3 (SEQ ID NO: 225): Position 3
- In HVR-L1 (SEQ ID NO: 226): positions 1, 3, 6, 11, 12, and 14
- In HVR-L2 (SEQ ID NO: 227): positions 1, 3, 4, and 7
-In HVR-L3 (SEQ ID NO: 228): positions 1 and 10

In certain embodiments, the substitutions provided herein are conservative substitutions. In certain embodiments, any one or more of the following substitutions may be made in any combination:
-In HVR-H1 (SEQ ID NO: 100): H2A, R or K
-In HVR-H2 (SEQ ID NO: 101): S4T; R5Q; G7H; D13E or R; K16R
-In HVR-H3 (SEQ ID NO: 102): K3A
-In HVR-L1 (SEQ ID NO: 113): T1D, Q or E; T3P; D6G; N11T; Y12W; S14H
-In HVR-L2 (SEQ ID NO: 114): E1F or Y; S3I; K4S; S7E or K
-In HVR-L3 (SEQ ID NO: 115): S1Q; M10T

All possible combinations of the above substitutions are SEQ ID NOs: 223, 224, 225, 226, 227 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively. , and encompassed by 228 consensus sequences.

In any of the above embodiments, the anti-CTLA-4 antibody is humanized. In one embodiment, the anti-CTLA-4 antibody comprises the HVR of any of the embodiments described above and further comprises an acceptor human framework (eg, a human immunoglobulin framework or a human consensus framework). In another embodiment, the anti-CTLA-4 antibody comprises the HVR of any of the above embodiments and further comprises a VH or VL comprising a FR sequence. In a further embodiment, the anti-CTLA-4 antibody comprises the following heavy chain and/or light chain variable domain FR sequences: For the heavy chain variable domain, FR1 has the amino acid sequence of any one of SEQ ID NOs: 229-232. FR2 includes the amino acid sequence of SEQ ID NO: 233, FR3 includes the amino acid sequence of SEQ ID NO: 234, and FR4 includes the amino acid sequence of SEQ ID NO: 235. For the light chain variable domain, FR1 comprises any one amino acid sequence of SEQ ID NOs: 236-238, FR2 comprises any one amino acid sequence of SEQ ID NOs: 240-241, and FR3 comprises any one of SEQ ID NOs: 242-241. FR4 contains any one amino acid sequence of SEQ ID NO: 245 to 246.

In another aspect, the anti-CTLA-4 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Contains heavy chain variable domain (VH) sequences with 99% or 100% sequence identity. In certain embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence , substitutions (eg, conservative substitutions), insertions, or deletions, an anti-CTLA-4 antibody containing the sequence retains the ability to bind CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and/or deleted in SEQ ID NO: 10. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, within the FR). Optionally, the anti-CTLA-4 antibody comprises the VH sequence in SEQ ID NO: 10, including post-translational modifications of that sequence. In certain embodiments, the VH is (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and (c) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 102. comprising one, two, or three HVRs selected from HVR-H3 comprising an amino acid sequence. Post-translational modifications include, but are not limited to, modification of the heavy chain or light chain N-terminus with glutamine or glutamic acid to pyroglutamic acid by pyroglutamylation.

In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 11. Anti-CTLA-4 antibodies are provided that include light chain variable domains (VL) with sequence identity. In certain embodiments, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence are , substitutions (eg, conservative substitutions), insertions, or deletions, an anti-CTLA-4 antibody containing the sequence retains the ability to bind CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and/or deleted in SEQ ID NO: 11. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, within the FR). Optionally, the anti-CTLA-4 antibody comprises the VL sequence in SEQ ID NO: 11, including post-translational modifications of that sequence. In certain embodiments, the VL is (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 115. comprising one, two, or three HVRs selected from HVR-L3 comprising an amino acid sequence. Post-translational modifications include, but are not limited to, modification of the heavy chain or light chain N-terminus with glutamine or glutamic acid to pyroglutamic acid by pyroglutamylation.

In another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 149. Anti-CTLA-4 antibodies are provided that include light chain variable domains (VL) with sequence identity. In certain embodiments, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence are , substitutions (eg, conservative substitutions), insertions, or deletions, an anti-CTLA-4 antibody containing the sequence retains the ability to bind CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and/or deleted in SEQ ID NO: 149. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (ie, within the FR). Optionally, the anti-CTLA-4 antibody comprises the VL sequence in SEQ ID NO: 149, including post-translational modifications of that sequence. In certain embodiments, the VL is (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 133. 1, 2, or 3 HVRs selected from HVR-L3 comprising an amino acid sequence. Post-translational modifications include, but are not limited to, modification of the heavy chain or light chain N-terminus with glutamine or glutamic acid to pyroglutamic acid by pyroglutamylation.

In another aspect, an anti-CTLA-4 antibody is provided comprising a VH in any of the embodiments described above, and a VL in any of the embodiments described above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 98 and SEQ ID NO: 99, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 97, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 86 and SEQ ID NO: 134, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 136 and SEQ ID NO: 95, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of the sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 136 and SEQ ID NO: 149, respectively, including post-translational modifications of the sequences. In a further aspect, a heterogeneous anti-CTLA-4 antibody is provided that comprises at least two different variable regions selected from among the variable regions comprising the VH and VL sequences described above. In one embodiment, the antibody comprised the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, with post-translational modifications of the sequences. Including, including things. In one embodiment, the antibody comprised the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, with post-translational modifications of the sequences. Including, including things. Post-translational modifications include, but are not limited to, modification of the heavy chain or light chain N-terminus with glutamine or glutamic acid to pyroglutamic acid by pyroglutamylation.

When the N-terminal amino acid of the heavy chain or light chain of the anti-CTLA-4 antibody provided herein is glutamine, the amino acid may be substituted with glutamic acid. When the N-terminal amino acid of the heavy chain or light chain of the anti-CTLA-4 antibody provided herein is glutamic acid, the amino acid may be substituted with glutamine.

In a further aspect, the invention provides antibodies that bind to the same epitope as the anti-CTLA-4 antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitope as the antibodies set forth in Table 4, Table 9, Table 14, and Table 19. In a specific embodiment, the third amino acid (Met), the 33rd amino acid (Glu), the 35th amino acid (Arg), the 53rd amino acid (Thr), the 97th amino acid (Glu) of SEQ ID NO: 28, 99th amino acid (Met), 100th amino acid (Tyr), 101st amino acid (Pro), 102nd amino acid (Pro), 103rd amino acid (Pro), 104th amino acid (Tyr), 105th amino acid Provided are antibodies that bind to an epitope in a fragment of CTLA-4 comprising at least one amino acid selected from the group consisting of amino acid (Tyr), and amino acid 106 (Leu). In certain embodiments, antibodies are provided that bind to an epitope in a fragment of CTLA-4 consisting of amino acids 97 (Glu) to 106 (Leu) of SEQ ID NO: 28. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of CTLA-4 consisting of amino acids 99 (Met) to 106 (Leu) of SEQ ID NO: 28.

In a further aspect of the invention, the anti-CTLA-4 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-CTLA-4 antibody is an antibody fragment, eg, an Fv, Fab, Fab', scFv, diabody, or F(ab') ₂ fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 antibody, a complete IgG4 antibody, or other antibody class or isotype as defined herein.

In a further aspect, the anti-CTLA-4 antibodies of the invention include an Fc region. In a further aspect, the anti-CTLA-4 antibodies of the invention include a constant region. The constant region may be a heavy chain constant region (including the Fc region), a light chain constant region, or both. In some embodiments, the Fc region is a native sequence Fc region. Exemplary heavy chain constant regions derived from natural antibodies include, for example, human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), human IgG4 (SEQ ID NO: 252). ) and other heavy chain constant regions. Other exemplary heavy chain constant regions include heavy chain constant regions such as SEQ ID NO: 82 and SEQ ID NO: 158. Exemplary light chain constant regions derived from natural antibodies include, for example, human κ chain (SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 159), human λ chain (SEQ ID NO: 53, SEQ ID NO: 87) Examples include light chain constant regions such as.

In another embodiment, the Fc region is a mutant Fc region created by adding amino acid modifications to the native sequence Fc region. In a specific embodiment, the mutant Fc region has enhanced binding activity for at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa, as compared to the native sequence Fc region. In a further embodiment, the mutant Fc region has enhanced binding activity for FcγRIIa and FcγRIIIa compared to the native sequence Fc region. Examples of heavy chain constant regions comprising such variant Fc regions include, for example, the heavy chain constant regions listed in Tables 26 to 30, SEQ ID NOs: 31, 32, 41 to 46, 65, 66, 81, 207, 239 , 253-271, 276, 277, 278, and the like.

The native sequence Fc region is usually organized as a homodimer consisting of two identical polypeptide chains. In certain embodiments, the variant Fc region may be a homodimer composed of polypeptide chains with the same sequence, or a heterodimer composed of polypeptide chains with mutually different sequences. good. Similarly, the heavy chain constant region including the Fc region may be a homodimer composed of polypeptide chains with the same sequence, or a heterodimer composed of polypeptide chains with mutually different sequences. There may be. Examples of heterologous heavy chain constant regions include, for example, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 31 and a polypeptide chain of SEQ ID NO: 32, a polypeptide chain of SEQ ID NO: 43 and a polypeptide chain of SEQ ID NO: 44. A heavy chain constant region comprising a peptide chain, a polypeptide chain of SEQ ID NO: 45 and a polypeptide chain of SEQ ID NO: 46, a polypeptide chain of SEQ ID NO: 254 and a polypeptide chain of SEQ ID NO: 256 A heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 257 and a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 258, a polypeptide chain of SEQ ID NO: 259 and a polypeptide chain of SEQ ID NO: 260 a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 261 and a polypeptide chain of SEQ ID NO: 263; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 262 and a polypeptide chain of SEQ ID NO: 264; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 265 and a polypeptide chain of SEQ ID NO: 267; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 266 and a polypeptide chain of SEQ ID NO: 268; , a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 269 and a polypeptide chain of SEQ ID NO: 270, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 271 and a polypeptide chain of SEQ ID NO: 81, Sequence A heavy chain constant region comprising a polypeptide chain of NO: 65 and a polypeptide chain of SEQ ID NO: 66, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 239 and a polypeptide chain of SEQ ID NO: 207, SEQ ID NO: A heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 259 and a polypeptide chain of SEQ ID NO: 276, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 65 and a polypeptide chain of SEQ ID NO: 278, etc. .

In a further aspect, an anti-CTLA-4 antibody according to any of the embodiments described above may incorporate any of the features described in items 1-7 below, alone or in combination.

1. Antibody Binding Activity In certain embodiments, the binding activity of the antibodies provided herein is ≦10 μM, ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, A dissociation constant (KD) of ≦0.01 nM, or ≦0.001 nM (eg, 10 ^-8 M or less, eg, 10 ^-8 M to 10 ^-13 M, eg, 10 ^-9 M to 10 ^-13 M).

In one embodiment, the binding activity of the antibody is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the in-solution binding affinity of a Fab for antigen can be determined by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I) labeled antigen in the presence of an increasing series of unlabeled antigen, and then coating the bound antigen with anti-Fab antibody. (See, eg, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated with 5 μg/ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) overnight. This is followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM of [ ¹²⁵ I]-antigen (e.g. anti-VEGF antibody, Fab- in Presta et al., Cancer Res. 57: 4593-4599 (1997) 12) Mix with serial dilutions of the Fab of interest. The Fab of interest is then incubated overnight, although this incubation can be continued for a longer period of time (eg, about 65 hours) to ensure that equilibrium is achieved. The mixture is then transferred to a capture plate for incubation (eg, 1 hour) at room temperature. The solution is then removed and the plate washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, 150 μl/well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximal binding is selected for use in competitive binding assays.

In one embodiment, the binding activity of the antibody is measured using a surface plasmon resonance analysis method, for example, using BIACORE (trademark) T200 or BIACORE (trademark) 4000 (GE Healthcare, Uppsala, Sweden). A ligand capture method is used. BIACORE (registered trademark) Control Software is used to operate the equipment. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions to attach a carboxymethyl dextran-coated sensor chip (GE Healthcare, Uppsala, Sweden) to a ligand capture molecule, e.g. Immobilize tag antibodies, anti-IgG antibodies, protein A, etc. Ligand capture molecules are diluted using a 10 mM sodium acetate solution at the appropriate pH and injected at the appropriate flow rate and injection time. For binding activity measurements, a buffer containing 0.05% polysorbate 20 (other name: Tween (trademark)-20) is used as the measurement buffer, flow rate is 10-30 μL/min, and measurement temperature is preferably 25°C or higher. Measured at 37°C. When performing a measurement by capturing an antibody as a ligand with a ligand capture molecule, after injecting the antibody and capturing the desired amount, serially dilute the antigen or Fc receptor prepared using the measurement buffer. A substance (analyte) is injected. When performing a measurement by capturing an antigen or Fc receptor as a ligand with a ligand capture molecule, inject the antigen or Fc receptor to capture the target amount, and then use a buffer prepared using a measurement buffer. Serial dilutions of antibody (analyte) are injected.

In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. Calculation of kinetics parameters is carried out by simultaneously fitting the binding and dissociation sensorgrams using a 1:1 Binding model, and calculates the binding rate (kon or ka), dissociation rate (koff or kd), the equilibrium dissociation constant (KD) can be calculated. If the binding activity is weak, especially if dissociation is rapid and calculation of kinetic parameters is difficult, the steady state model may be used to calculate the equilibrium dissociation constant (KD). Other parameters of binding activity include dividing the amount of analyte bound (RU) at a specific concentration by the captured amount of ligand (RU) to calculate the "amount of analyte bound per unit amount of ligand". good.

2. Antibody Fragments In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); in addition, WO93/ 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab') ₂ fragments containing salvage receptor binding epitope residues with increased in vivo half-lives.

Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. For example, EP404,097; WO1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993 ) reference. Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

Single domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, eg, US Pat. No. 6,248,516 B1).

Antibody fragments can be produced in a variety of ways, including, but not limited to, proteolytic digestion of whole antibodies, production by recombinant host cells (e.g., E. coli or phage), as described herein. It can be made using the following method.

3. Chimeric and Humanized Antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (eg, a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies contain one or more variable domains in which HVRs (e.g. CDRs (or portions thereof)) are derived from non-human antibodies and FRs (or portions thereof) are derived from human antibody sequences. Originates from Humanized antibodies optionally include at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are removed from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody. ) with the corresponding residue from

Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and also, for example, Riechmann et al., Nature 332: 323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28: 489-498 (1991) (describing resurfacing); Dall'Acqua et al., Methods 36: 43-60 (2005) (describing FR shuffling); and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252 -260 (2000) (describing a "guided selection" approach for FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to: Framework regions; framework regions derived from human antibody consensus sequences of specific subgroups of light or heavy chain variable regions (Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992) and Presta et al. J. Immunol., 151: 2623 (1993)); human mature (somatic mutation) framework regions or human germline framework regions (e.g., Almagro and Fransson, Front. Biosci. 13: 1619 -1633 (2008)); and framework regions derived from screening of FR libraries (Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

4. Human Antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by a variety of techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

Human antibodies may be prepared by administering an immunogen to transgenic animals that have been modified to produce fully human antibodies or complete antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, where all or a portion of the human immunoglobulin loci replaces endogenous immunoglobulin loci or is extrachromosomally or It exists randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Pat. No. 7,041,870; and also U.S. Patent Application Publication No. 2007/0061900, which describes VELOCIMOUSE® technology. Human variable regions from complete antibodies produced by such animals may be further modified, eg, by combining with different human constant regions.

Human antibodies can also be made using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been previously described (e.g. Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production). Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include, for example, U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, - human hybridomas). Human hybridoma technology (trioma technology) is also discussed in Vollmers and Brandlein, Histology and Histopathology, 20(3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-191. (2005).

Human antibodies can also be produced by isolating selected Fv clone variable domain sequences from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. A method for selecting human antibodies from an antibody library will be described below.

5. Library-Derived Antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further, e.g., McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al. , J. Immunol. Methods 284(1-2): 119-132 (2004).

In a particular phage display method, a repertoire of VH and VL genes is cloned separately by polymerase chain reaction (PCR) and recombined in a random phage library, which is described by Winter et al. ., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, a naive repertoire can be cloned (e.g., from humans) to provide a wide range of non-self and It can also provide a single source of antibodies to self-antigens. Finally, a naive library was created by cloning unrearranged V-gene segments from stem cells and generating hypervariable CDR3 as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). It can also be made synthetically by using PCR primers that encode the region and contain random sequences to accomplish rearrangement in vitro. Patent documents describing human antibody phage libraries include, for example: U.S. Pat. Including number /0160598, number 2007/0237764, number 2007/0292936 and number 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered herein to be human antibodies or human antibody fragments.

6. Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (eg, bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for CTLA-4 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CTLA-4. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing CTLA-4. Bispecific antibodies can be prepared as full-length antibodies or as antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (Milstein and Cuello, Nature 305: 537 (1983), WO93/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole technology (see, eg, US Pat. No. 5,731,168). Multispecific antibodies manipulate electrostatic steering effects to create Fc heterodimeric molecules (WO2009/089004A1); crosslinking two or more antibodies or fragments (U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to create antibodies with dual specificity (Kostelny et al., J. Immunol., 148(5) ): 1547-1553 (1992)); generating bispecific antibody fragments using "diabody" technology (Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see Gruber et al., J. Immunol., 152: 5368 (1994)); and, for example, Tutt et al. J. Immunol. 147:60 (1991) by preparing trispecific antibodies.

Also included herein are engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" (see, eg, US Patent Application Publication No. 2006/0025576 A1).

Antibodies or fragments herein also include "dual-acting Fabs" or "DAFs" that contain one antigen-binding site that binds CTLA-4 and another distinct antigen (e.g., U.S. Pat. /0069820).

7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (eg, antigen binding).

a) Substitution, Insertion, and Deletion Variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into antibodies of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. good.

Amino acids can be divided into groups by common side chain properties:
(1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile);
(2) Neutral hydrophilicity: cysteine (Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln);
(3) Acidic: aspartic acid (Asp), glutamic acid (Glu);
(4) Basic: histidine (His), lysine (Lys), arginine (Arg);
(5) Residues that affect chain orientation: glycine (Gly), proline (Pro);
(6) Aromaticity: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).
Non-conservative substitutions refer to exchanging a member of one of these classes for another.

One type of substitutional variant involves substitution of one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Typically, the resulting variants selected for further study are modified (e.g., improved) in certain biological properties compared to the parent antibody (e.g., increased affinity, decreased immunogenicity). ) and/or will substantially retain certain biological properties of the parent antibody. Exemplary substitutional variants are affinity matured antibodies, which can be appropriately generated using, for example, phage display-based affinity maturation techniques (eg, as described herein). Briefly, one or more HVR residues are mutated and the mutated antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity).

Modifications (eg, substitutions) can be made in the HVR, eg, to improve antibody affinity. Such modifications may affect HVR "hotspots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and/or in residues that contact the antigen, and the resulting mutated VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from a secondary library can be performed, e.g., Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001 )) It is described in. In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by any of a variety of methods (eg, error-prone PCR, chain shuffling or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves HVR-directed approaches that randomize several HVR residues (eg, 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs so long as such modifications do not substantially reduce the ability of the antibody to bind antigen. For example, conservative modifications (eg, conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the HVR. Such modifications can be, for example, outside of the antigen-contacting residues of the HVR. In certain embodiments of the mutant VH and VL sequences described above, each HVR is unaltered or contains only one, two, or three amino acid substitutions.

A useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244: 1081-1085. It is called. In this method, a residue or group of target residues (e.g., charged residues such as arginine, aspartate, histidine, lysine, and glutamate) are identified and neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Additional substitutions may be introduced at amino acid positions that have shown functional sensitivity to this initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex may be analyzed to identify points of contact between the antibody and the antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or may be excluded from substitution candidates. Variants can be screened to determine whether they contain desired properties.

Insertions of amino acid sequences, as well as insertions of single or multiple amino acid residues within a sequence, can range in length from one to 100 or more residues of a polypeptide at the amino and/or carboxyl terminus. Including fusion. Examples of terminal insertions include antibodies with a methionyl residue at the N-terminus. Other insertional variants of antibody molecules include those fused to the N- or C-terminus of the antibody with an enzyme (eg, for ADEPT) or a polypeptide that increases the plasma half-life of the antibody.

b) Glycosylation Variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites.

If the antibody includes an Fc region, the carbohydrates attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are usually attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15: 26-32 (1997). Oligosaccharides include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of oligosaccharides in antibodies of the invention may be performed to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is the sum of all sugar structures (e.g. complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry as described in e.g. WO2008/077546. It is determined by calculating the average amount of fucose in the sugar chain at Asn297. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence diversity among antibodies, Asn297 may be located ±3 amino acids upstream or downstream of position 297, ie, between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, eg, US Patent Application Publication Nos. 2003/0157108 (Presta, L.); 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications regarding "defucosylated" or "fucose-deficient" antibody variants are US2003/0157108; WO2000/61739; WO2001/29246; US2003/0115614; US2002/0164328; US2004/0093621; US2004/0132140; 4/ WO2003/084570; WO2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Ok azaki et al. J. Mol. Biol. 336: 1239-1249 ( 2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). An example of a cell line that can produce defucosylated antibodies is Lec13 CHO cells, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Pat. US2003/0157108A1, Presta, L; and WO2004/056312A1, Adams et al., especially Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (e.g. Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003/085107). .

Further provided are antibody variants having bisected oligosaccharides, for example, where a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US2005/0123546 (Umana et al.) There is. Antibody variants having at least one galactose residue in the oligosaccharide added to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997/30087 (Patel et al.); WO1998/58964 (Raju, S.); and WO1999/22764 (Raju, S.).

c) Fc Region Variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. Fc region variants may include human Fc region sequences (eg, human IgG1, IgG2, IgG3, or IgG4 Fc regions) that include amino acid modifications (eg, substitutions) at one or more amino acid positions.

In certain embodiments, antibody variants that possess some, but not all, effector functions are also within the contemplation of the present invention, where the effector functions make the antibody unique, although its in vivo half-life is important. This makes them desirable candidates for applications where certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity measurements can be performed to confirm the reduction/deficiency of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding measurements can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while maintaining FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). A non-limiting example of an in vitro assay for assessing ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); US Pat. No. 5,821,337 (Bruggemann, M. et al. , J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays may be used (e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay. assays (Promega, Madison, WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). may be done. C1q binding assays may also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISA in WO2006/029879 and WO2005/100402. CDC measurements may also be performed to assess complement activation (e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al., Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103: 2738-2743 (2004)). Additionally, determination of FcRn binding and in vivo clearance/half-life may also be performed using methods known in the art (e.g. Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

Antibodies with reduced effector function include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737,056). Such Fc variants include amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant (U.S. Pat. No. 7,332,581) with substitutions of residues 265 and 297 to alanine. Includes Fc variants with two or more substitutions.

Certain antibody variants with increased or decreased binding to FcRs have been described (US Pat. No. 6,737,056; WO2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, the antibody variant has one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and/or 334 (residues in EU numbering) of the Fc region). Contains the associated Fc region.

In some embodiments, it has been modified (i.e., increased or Modifications are made in the Fc region that result in C1q binding (which is either decreased) and/or complement dependent cytotoxicity (CDC).

increased half-life, and neonatal Fc receptors (FcRn: responsible for transferring maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976); Kim et al., J. Immunol. 24: 249 (1994))) are described in US Patent Application Publication No. 2005/0014934 A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions therein that increase the binding of the Fc region to FcRn. Such Fc variants include Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382 , 413, 424, or 434 (eg, substitution of Fc region residue 434 (US Pat. No. 7,371,826)).

For other examples of Fc region variants, see also Duncan & Winter, Nature 322: 738-740 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO94/29351.

d) Cysteine Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine engineered antibodies (eg, "thioMAbs") in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the residues that are substituted occur at accessible sites on the antibody. By replacing those residues with cysteine, a reactive thiol group is placed at an accessible site on the antibody, and the reactive thiol group is attached to another moiety (drug moiety or linker-drug moiety). etc.) may be used to create immunoconjugates as described in further detail herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region. (EU numbering). Cysteine-engineered antibodies may be produced, for example, as described in US Pat. No. 7,521,541.

e) Antibody Derivatives In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties that are known and readily available in the art. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly1,3 Dioxolane, poly 1, 3, 6 trioxane, ethylene/maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing because of its stability to water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. Generally, the number and/or type of polymers used for derivatization will depend on the particular property or function of the antibody to be improved, including, but not limited to, the ability of the antibody derivative to improve under defined conditions. The decision can be made based on considerations such as whether it will be used for therapy or not.

In another embodiment, conjugates of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, heating the non-protein portion to a temperature that does not harm normal cells but kills cells in close proximity to the antibody-non-protein portion. Contains wavelength.

B. Recombinant Methods and Constructions Antibodies can be produced using recombinant methods and constructions, for example, as described in US Pat. No. 4,816,567. In one aspect, isolated nucleic acids encoding anti-CTLA-4 antibodies described herein are provided. Such nucleic acids may encode amino acid sequences comprising the VL and/or VH of the antibody (eg, the light chain and/or heavy chain of the antibody). In further embodiments, one or more vectors (eg, expression vectors) containing such nucleic acids are provided. In further embodiments, host cells containing such nucleic acids are provided. In one such embodiment, the host cell contains a vector comprising a nucleic acid encoding (1) an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) an amino acid sequence comprising the VL of the antibody. A first vector comprising a nucleic acid encoding a sequence and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody (eg, has been transformed). In one embodiment, the host cell is eukaryotic (eg, a Chinese hamster ovary (CHO) cell) or a lymphoid cell (eg, a Y0, NS0, Sp2/0 cell). In one embodiment, culturing a host cell comprising a nucleic acid encoding an anti-CTLA-4 antibody as described above under conditions suitable for expression of an anti-CTLA-4 antibody, and optionally injecting the antibody into the host cell (or host cell culture). A method of producing an anti-CTLA-4 antibody is provided, the method comprising recovering an anti-CTLA-4 antibody from a culture medium.

For recombinant production of anti-CTLA-4 antibodies, a nucleic acid encoding the antibody (such as those described above) is isolated and one or more antibodies are isolated for further cloning and/or expression in host cells. Insert into vector. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of antibodies). by using).

Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, especially if glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. See also Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp.245-254). After expression, antibodies may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, bacteria such as filamentous fungi or yeast, including strains of fungi and yeast whose glycosylation pathways have been "humanized", resulting in the production of antibodies with a partially or fully human glycosylation pattern. Nuclear microorganisms are preferred cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

Those derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that are used for conjugation with insect cells, particularly for the transformation of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., US Pat.

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension would be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 strain (COS-7); the human embryonic kidney strain (Graham et al., J. Gen Virol. 36: 59 (1977 ); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, described in Mather, Biol. Reprod. 23: 243-251 (1980), etc.); monkey kidney cells (CV1 ); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); dog kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes ( Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (eg, described in Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines are Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and Y0, Includes myeloma cell lines such as NS0, and Sp2/0. For a review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268. (2003).

Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. The relevant antigen is combined with a protein that is immunogenic in the species to be immunized, e.g. keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, with a bifunctional substance or derivatizing agent, e.g. , maleimidobenzoyl sulfosuccinimide ester (conjugation via a cysteine residue), N-hydroxysuccinimide (via a lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂ , or R ¹ N=C=NR (where R and R ¹ are different alkyl groups).

Animals (usually non-human mammals) can be treated, for example, by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally at multiple sites. immunization against an antigen, immunogenic conjugate, or derivative. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. After 7-14 days, the animals are bled and the serum is assayed for antibody titers. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with a conjugate of the same antigen, but conjugated to a different protein and/or conjugated via a different cross-linking reagent. Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, a flocculant such as alum is also preferably used to enhance the immune response.

Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies that constitute the population are free from naturally occurring potential mutations and/or post-translational modifications (e.g., Identical except for isomerization and amidation). Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of distinct antibodies.

For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature 256(5517): 495-497 (1975). In hybridoma methods, mice or other suitable host animals, such as hamsters, are immunized as described herein above to produce or be capable of producing antibodies that specifically bind to the protein used for immunization. induce certain lymphocytes. Alternatively, lymphocytes can be immunized in vitro.

The immunizing agent typically includes the antigenic protein or fusion variant thereof. Generally, peripheral blood lymphocytes (PBL) are used when cells of human origin are desired, and spleen cells or lymph node cells are used when a non-human mammalian source is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59- 103).

Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually rat or mouse myeloma cell lines are utilized. The hybridoma cells thus produced are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, a substance that inhibits the growth of HGPRT-deficient cells, and thymidine (HAT medium).

Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. . Among these, mouse myeloma strains, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and from the American Type Culture Collection, Manassas, VA, USA SP-2 cells (and derivatives thereof, such as X63-Ag8-653) are preferred. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., J Immunol. 133(6): 3001-3005 (1984); Brodeur et al. ., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

The culture medium in which hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and measurements are known in the art. For example, binding affinity can be determined by Scatchard analysis of Munson, Anal Biochem. 107(1): 220-239 (1980).

Once hybridoma cells producing antibodies of the desired specificity, affinity, and/or activity are identified, the clones can be subcloned by limiting dilution and propagated by standard methods (Goding, supra). . Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors within mammals.

Monoclonal antibodies secreted by the subclones are purified from culture medium, ascites, or serum by conventional immunoglobulin purification methods, e.g., protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. , properly separated.

Antibodies may be produced by immunizing a suitable host animal against the antigen. In one embodiment, the antigen is a polypeptide comprising full-length CTLA-4. In one embodiment, the antigen is a polypeptide comprising soluble CTLA-4. In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acid 97 (Glu) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acids 99 (Met) to 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). The invention also includes antibodies produced by immunizing an animal against the antigen. The antibody may incorporate any of the features described in the exemplary anti-CTLA-4 antibodies described above, alone or in combination.

C. Measurement method (assay)
Anti-CTLA-4 antibodies provided herein can be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art. may be made into

1. Binding and Other Assays In one aspect, antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, surface plasmon resonance assay, and the like.

In another aspect, competes with an anti-CTLA-4 antibody described herein (e.g., an anti-CTLA-4 antibody described in Table 4, Table 9, Table 14, and Table 19) for binding to CTLA-4. Competition assays can be used to identify antibodies that. In certain embodiments, when such competing antibodies are present in excess, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, or more, binding of the reference antibody to CTLA-4 is inhibited (e.g., reduced). In some instances, binding is inhibited by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such competing antibodies are bound by anti-CTLA-4 antibodies described herein (e.g., anti-CTLA-4 antibodies described in Table 4, Table 9, Table 14, and Table 19). binds to the same epitope (e.g., a linear or conformational epitope) that it binds to. Detailed exemplary methods for mapping epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, immobilized CTLA-4 is tested for a first labeled antibody that binds to CTLA-4 and for its ability to compete with the first antibody for binding to CTLA-4. A second is incubated in a solution containing unlabeled antibody. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CTLA-4 is incubated in a solution containing a first labeled antibody but no second unlabeled antibody. After incubation under conditions that permit binding of the first antibody to CTLA-4, excess unbound antibody is removed and the amount of label bound to immobilized CTLA-4 is measured. If the amount of label bound to immobilized CTLA-4 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is not as strong as the first antibody in binding to CTLA-4. Indicates a conflict. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity Assay In one aspect, an assay for identifying biologically active anti-CTLA-4 antibodies is provided. Biological activities may include, for example, cell proliferation inhibitory activity, cytotoxic activity (eg, ADCC/CDC activity, ADCP activity), immunostimulatory activity, CTLA-4 inhibitory activity. Also provided are antibodies having such biological activity in vivo and/or in vitro.

In certain embodiments, antibodies of the invention are tested for such biological activity.

In certain embodiments, antibodies of the invention are tested for their ability to inhibit cell growth or proliferation in vitro. Methods for measuring inhibition of cell growth or proliferation are well known in the art. Certain cell proliferation assays, exemplified by the "cell killing" assay described herein, measure cell viability. One such assay is the CellTiter-Glo™ Luminescent Cell Viability Assay, commercially available from Promega (Madison, Wis.). This assay determines the number of living cells in a culture based on the abundance of ATP, an indicator of metabolically active cells. See Crouch et al (1993) J. Immunol. Meth. 160: 81-88, US Pat. No. 6,602,677. The assay may be performed in a 96- or 384-well format, making it amenable to automated high-throughput screening (HTS). See Cree et al (1995) AntiCancer Drugs 6: 398-404. The assay procedure involves adding a single reagent (CellTiter-Glo® reagent) directly to cultured cells. This lyses the cells and a luciferase reaction produces a luminescent signal. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of live cells present in the culture. Data may be recorded by a luminometer or a CCD camera imager. The luminescence value is expressed by relative light unit (RLU).

Another cell proliferation assay is the ``MTT'', a colorimetric assay that measures the oxidation of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide to formazan by mitochondrial reductase. ” assay. Similar to the CellTiter-Glo™ assay, this measurement indicates the number of metabolically active cells present in a cell culture. See, eg, Mosmann (1983) J. Immunol. Meth. 65: 55-63 and Zhang et al. (2005) Cancer Res. 65: 3877-3882.

Cells used in any of the above in vitro assays include cells or cell lines that naturally express CTLA-4 or that have been engineered to express CTLA-4. Such cells also include cell lines that express CTLA-4 and cell lines that do not normally express CTLA-4 but have been transfected with a nucleic acid encoding CTLA-4.

In one aspect, anti-CTLA-4 antibodies are tested for their ability to inhibit cell growth or proliferation in vivo. In certain embodiments, anti-CTLA-4 antibodies are tested for their ability to inhibit tumor growth in vivo. In vivo model systems such as xenograft models can be used for such testing. In an exemplary xenograft system, human tumor cells are introduced into an appropriately immunodeficient non-human animal, such as an athymic "nude" mouse. An antibody of the invention is administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is determined. In certain embodiments of the xenograft systems described above, the human tumor cells are tumor cells derived from a human patient. Such xenograft models are commercially available from Oncotest GmbH (Frieberg, Germany). In certain embodiments, human tumor cells are introduced into an appropriately immunocompromised non-human animal by subcutaneous injection or by implantation into a suitable site, such as the mammary fat pad.

It is understood that any of the above assays can be performed using the immunoconjugates of the invention instead of or in addition to anti-CTLA-4 antibodies.

A typical assay for measuring ADCC activity of therapeutic antibodies is based on a ⁵¹ Cr release assay and involves the following steps: labeling target cells with [ ⁵¹ Cr]Na ₂ CrO ₄ ; cell surface opsonizing the target cells expressing the antigen thereon with the antibody; combining the opsonized radiolabeled target cells and effector cells in a suitable ratio in a microtiter plate in the presence or absence of the test antibody; ; incubating the mixture of cells for preferably 16-18 hours, preferably at 37°C; collecting the supernatant; and analyzing the radioactivity in the supernatant sample. The cytotoxicity of the test antibody is then determined, for example, by the following formula: Specific cytotoxicity (%) = (Radioactivity in the presence of antibody - Radioactivity in the absence of antibody) / (Maximum radioactivity - Radioactivity in the absence of antibody) x 100. Graphs can be generated by varying the target cell:effector cell ratio or antibody concentration.

To assess complement activation, a complement-dependent cytotoxicity (CDC) assay was performed, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). can do. Briefly, varying concentrations of the polypeptide variant and human complement are diluted in buffer. Cells expressing the antigen to which the polypeptide variant binds are diluted to a density of ˜1×10 ⁶ cells/ml. The mixture of polypeptide variants, diluted human complement, and antigen-expressing cells is added to a flat bottom tissue culture 96-well plate and allowed to incubate for 2 hours at 37°C and 5% _CO2 to promote complement-mediated cell lysis. 50 μl of Alamar Blue (Accumed International) is then added to each well and incubated overnight at 37°C. Measure absorbance using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. Results are expressed in relative fluorescence units (RFU). Sample concentrations can be calculated from the standard curve and the percent activity compared to the non-variant polypeptide is reported for the polypeptide variant of interest.

An exemplary assay method for ADCP activity can include: coating target bioparticles, such as E. coli-labeled FITC (Molecular Probes) or Staphylococcus aureus-FITC, with the test antibody; forming opsonized particles. the opsonized particles described above were added to THP-1 effector cells (a monocytic cell line, available from ATCC) at a ratio of 1:1, 10:1, 30:1, 60:1, 75:1, or 100:1. The cells are preferably incubated with E. coli-FITC/antibody for 1.5 hours at 37°C; after incubation, trypan blue is added to the cells (preferably for 2 hours at room temperature) to generate FcγR-mediated phagocytosis. ~3 min) to quench the fluorescence of bacteria that are not internalized and adhere to the outside of the cell surface; transfer the cells into FACS buffer (e.g., 0.1% BSA, 0.1% sodium azide in PBS) and FACS Assay the fluorescence of THP-1 cells using a BD FACS Calibur (e.g., BD FACS Calibur). To assay the extent of ADCP, gates are preferably set on THP-1 cells and the median fluorescence intensity is measured. In a most preferred embodiment, the ADCP assay is performed using E. coli-FITC (control), E. coli-FITC and THP-1 cells (used as FcγR-independent ADCP activity), E. coli-FITC, THP-1 cells and test cells in culture medium. Performed using an antibody (used as FcγR-dependent ADCP activity).

Cytotoxic activity by antibodies usually involves binding of the antibody to the cell surface. Whether or not the antigen is expressed on the surface of the target cell can be appropriately confirmed by methods known to those skilled in the art such as FACS.

Immune activation can be detected using a cellular immune response or a humoral immune response as an indicator. Specifically, increased expression of cytokines (e.g., IL-6, G-CSF, IL-12, TNFα, and IFNγ) or their receptors, and increased expression of immune cells (e.g., B cells, T cells, NK cells, etc.) , macrophages, monocytes, etc.), enhancement of activation state, hyperfunction, or enhancement of cytotoxic activity. In particular, T cell activation can be detected by measuring increased expression of activation markers such as CD25, CD69 and ICOS. For example, it is known that patients receiving the anti-CTLA-4 antibody ipilimumab have an increase in ICOS ⁺ CD4 ⁺ T cells in their peripheral blood after administration, and this is due to systemic administration of the anti-CTLA-4 antibody. This is thought to be an effect of activated immune status (Cancer Immunol. Res. (2013) 1(4): 229-234).

Activation of T cells requires not only antigen receptor (TCR)-mediated stimulation but also CD28-mediated co-stimulation. When CD28 on the surface of a T cell binds to B7-1 (CD80) or B7-2 (CD86) on the surface of an antigen-presenting cell, an auxiliary signal is transmitted into the T cell and the T cell is activated. . On the other hand, CTLA-4 is expressed on the surface of activated T cells. CTLA-4 binds to CD80 and CD86 with a stronger affinity than CD28, so it interacts with CD80 and CD86 preferentially than with CD28, resulting in suppression of T cell activation.

Based on such a mechanism of action, the inhibitory activity against CTLA-4 can be measured as the activity of inhibiting the binding between CTLA-4 and CD80 or CD86. In one embodiment, an assay for measuring inhibitory activity against CTLA-4 comprises the following steps: binding purified CTLA-4 protein to a support such as a microtiter plate or magnetic beads, a test antibody and a labeled soluble Adding CD80 or CD86, washing away unbound components, and quantifying bound labeled CD80 or CD86. Whether a test antibody cross-reacts with CD28 can be determined by performing a similar assay in which CD28 replaces CTLA-4. In another embodiment, the inhibitory activity against CTLA-4 can also be measured using a functional assay that detects T cell activation as described above. For example, in a system that stimulates a T cell population with cells expressing CD80 or CD86 and measures T cell activation, if a test antibody with CTLA-4 inhibitory activity is added, Further enhancement of activation results.

D. Immunoconjugates The present invention also includes one or more cytotoxic agents (e.g. chemotherapeutic or chemotherapeutic agents, growth inhibitors, toxins (e.g. protein toxins of bacterial, fungal, plant or animal origin, enzymatically active agents). Provided are immunoconjugates comprising an anti-CTLA-4 antibody herein conjugated to a toxin (or a radioactive isotope) or a radioactive isotope).

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to: There are: maytansinoids (see US Patent Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); e.g. monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (US Patents 5,635,483 and 5,780,588) and 7,498,298); dolastatin; calicheamicin or derivatives thereof (U.S. Pat. and No. 5,877,296; Hinman et al., Cancer Res. 53: 3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin ( Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16 : 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 ( 2002); King et al., J. Med. Chem. 45: 4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to: diphtheria A chain , non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii protein, diantin Proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitors, curcin, crotin, saponaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for making radioactive conjugates. Examples include radioisotopes of ²¹¹ At, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹² Bi, ³² P, ²¹² Pb and Lu. When a radioactive conjugate is used for detection, it can be used as a radioactive atom for scintigraphic examination (e.g. Tc-99m or ^123I ) or for nuclear magnetic resonance (NMR) imaging (magnetic resonance imaging, MRI). (also known as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) .

Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein linkers. For example, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters. (e.g. dimethyl adipimidate HCl), active esters (e.g. disuccinimidyl suberate), aldehydes (e.g. glutaraldehyde), bis-azido compounds (e.g. bis(p-azidobenzoyl)hexanediamine), bis- Diazonium derivatives (e.g. bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g. toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g. 1,5-difluoro-2,4-dinitrobenzene) It is. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionuclides to antibodies. See WO94/11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug within the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020) are used. obtain.

The immunoconjugates or ADCs herein include BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.); SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone) Expressly contemplated are conjugates prepared using cross-linking reagents including, but not limited to, benzoate).

E. Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the anti-CTLA-4 antibodies provided herein are useful for detecting the presence of CTLA-4 in a biological sample. The term "detection" as used herein encompasses quantitative or qualitative detection. In certain embodiments, the biological sample is a cell or tissue, such as serum, whole blood, blood sample, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, Includes ascites, milk, colostrum, mammary gland secretions, lymph, urine, sweat, tears, gastric fluid, joint fluid, ascites, eye fluid, or mucus.

In one embodiment, anti-CTLA-4 antibodies are provided for use in diagnostic or detection methods. In a further aspect, a method of detecting the presence of CTLA-4 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-CTLA-4 antibody described herein under conditions that permit binding of the anti-CTLA-4 antibody to CTLA-4; and It includes detecting whether a complex is formed between the anti-CTLA-4 antibody and CTLA-4. Such methods may be in vitro or in vivo methods. In one embodiment, anti-CTLA-4 antibodies are used to select subjects suitable for treatment with anti-CTLA-4 antibodies, eg, where CTLA-4 is a biomarker for patient selection.

The antibodies of the present invention can be used, for example, to check the state of immune response and diagnose immune system dysfunction.

In certain embodiments, labeled anti-CTLA-4 antibodies are provided. Labels include labels or moieties that are detected directly (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) as well as those that are detected indirectly, such as through enzymatic reactions or intermolecular interactions. including, but not limited to, moieties such as enzymes or ligands. Exemplary labels include, but are not limited to: radioisotopes ^32P , ^14C , ^125I , ^3H and ^131I , fluorophores such as rare earth chelates or fluorescein and its derivatives. , rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline Heterocyclic oxidases such as phosphatases, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g. glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), uricase and xanthine oxidase, dye precursors using hydrogen peroxide oxidizing enzymes (such as HRP, lactoperoxidase, or microperoxidase), biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations Pharmaceutical formulations of the anti-CTLA-4 antibodies described herein include the antibody having the desired purity in one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol , A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to: phosphates, citrates, buffers such as acid salts, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); small molecule (less than about 10 residues) polypeptides; serum albumin, gelatin, or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-containing agents such as polyethylene glycol (PEG). Ionic surfactant. Exemplary pharmaceutically acceptable carriers herein further include human soluble glycoproteins such as soluble neutral active hyaluronidase glycoprotein (sHASEGP) (e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.)). Contains interstitial drug dispersants such as PH-20 (hyaluronidase glycoprotein). Certain exemplary sHASEGPs and methods of their use (including rHuPH20) are described in US Patent Application Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO2006/044908, the latter formulation containing a histidine-acetate buffer.

The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated. Those with complementary activities that do not adversely affect each other are preferred. Such active ingredients are present in suitable combinations and in amounts effective for their intended purposes.

The active ingredient is incorporated into microcapsules (e.g. hydroxymethyl cellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively) prepared by e.g. droplet formation (coacervation) techniques or by interfacial polymerization. It can be incorporated into colloidal drug delivery systems (eg, liposomes, albumin spherules, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained release formulations may also be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films or microcapsules.

Preparations used for in vivo administration are usually sterile. Sterility is easily achieved, such as by filtration through a sterile filtration membrane.

G. Therapeutic Methods and Therapeutic Compositions Any of the anti-CTLA-4 antibodies provided herein may be used in therapeutic methods.
In one aspect, anti-CTLA-4 antibodies are provided for use as a pharmaceutical. In a further aspect, anti-CTLA-4 antibodies are provided for use in treating tumors. In certain embodiments, anti-CTLA-4 antibodies are provided for use in therapeutic methods. In certain embodiments, the invention provides an anti-CTLA-4 antibody for use in a method of treating an individual with a tumor, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody. I will provide a. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, as described below). In a further aspect, the invention provides anti-CTLA-4 antibodies for use in cell injury. In certain embodiments, the invention provides for use in a method of injuring cells in an individual, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody to injure the cells. Provides anti-CTLA-4 antibodies. In a further aspect, the invention provides anti-CTLA-4 antibodies for use in immune activation. In certain embodiments, the invention provides for use in a method of activating immunity in an individual, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody to activate immunity. provides anti-CTLA-4 antibodies. The "individual" according to any of the above embodiments is preferably a human.

In some embodiments, the tumor is a solid tumor. In solid tumors, tumor cells usually proliferate and form a population, and tumor tissue is mainly formed by these cells. Furthermore, in many cases, tumor tissue in vivo is infiltrated with immune cells such as lymphocytes, and these cells also constitute part of the tumor tissue. In some embodiments, the tumor tissue is infiltrated with immune cells, particularly regulatory T (Treg) cells. In one embodiment, damage to cells is caused by ADCC activity, CDC activity, or ADCP activity. In one embodiment, cells expressing CTLA-4 on their cell surface are injured. In further embodiments, the cells that are injured are Treg cells. In certain embodiments, Treg cells infiltrating within tumor tissue are damaged. In one embodiment, immunity is activated by damaging Treg cells (immune suppression by Treg cells is released). In a further embodiment, immunity (particularly anti-tumor immunity) in tumor tissue is activated. In some embodiments, the immune activation is T cell activation.

In a further aspect, the degree of pharmaceutical efficacy provided by the anti-CTLA-4 antibody of the present invention varies depending on the tissue within an individual. In certain embodiments, the extent varies depending on the concentration of adenosine-containing compounds within the tissue. In a further embodiment, the effect is increased in tissues with high concentrations of adenosine-containing compounds compared to tissues with low concentrations of adenosine-containing compounds. Examples of tissues with high concentrations of adenosine-containing compounds include tumor tissues. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues such as normal tissues. In some embodiments, immunity is activated more strongly in tumor tissue than in non-tumor tissue. Such differences in response need not be observed at all doses of anti-CTLA-4 antibody, but only at a certain range of doses. In another embodiment, immunity is activated at a lower dose in tumor tissue compared to non-tumor tissue. Also, in another embodiment, therapeutic effects are observed at lower doses than side effects are observed. In certain embodiments, a therapeutic effect is the development of an anti-tumor effect (e.g., tumor regression, induction of cell death or growth inhibition of tumor cells), and a side effect is an autoimmune disease (e.g., an autoimmune disease (an overactive immune response that causes damage to normal tissue). (including injury to).

In a further aspect, the degree of pharmaceutical efficacy provided by the anti-CTLA-4 antibody of the present invention is determined by an adenosine-containing compound-dependent (i.e., varying depending on the concentration of the adenosine-containing compound) binding activity to CTLA-4. It depends on whether you have it or not. In some embodiments, the anti-CTLA-4 antibodies of the invention are antibodies whose binding activity for CTLA-4 increases as the concentration of the adenosine-containing compound increases. In some embodiments, the control anti-CTLA-4 antibody is an antibody that does not have CTLA-4 binding activity that is dependent on the concentration of the adenosine-containing compound. In certain embodiments, an antibody that does not have a CTLA-4 binding activity that is dependent on the concentration of an adenosine-containing compound is one in which the difference in CTLA-4 binding activity in the presence and absence of the compound is, for example, more than 2-fold. means an antibody that is small, less than 1.8 times, less than 1.5 times, less than 1.3 times, less than 1.2 times, or less than 1.1 times. It is desirable that the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody have substantially the same CTLA-4 binding activity in the presence of a sufficient amount of an adenosine-containing compound.

In a specific aspect, the anti-CTLA-4 antibody of the present invention and a control anti-CTLA-4 antibody have different effects as pharmaceuticals brought about by each antibody. In certain embodiments, the pharmaceutical efficacy is different in tissues where the concentration of the adenosine-containing compound is low. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues such as normal tissues. Anti-CTLA-4 antibodies can also be provided as pharmaceutical formulations containing them. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, anti-CTLA-4 antibodies of the invention exhibit lower levels of immune activation compared to control anti-CTLA-4 antibodies. In some embodiments, in tissues with lower concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the invention can be administered at lower doses required to activate immunity compared to control anti-CTLA-4 antibodies. is high. In some embodiments, anti-CTLA-4 antibodies of the invention exhibit reduced levels of side effects compared to control anti-CTLA-4 antibodies in tissues with low concentrations of adenosine-containing compounds. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, anti-CTLA-4 antibodies of the invention require higher doses at which side effects are observed compared to control anti-CTLA-4 antibodies. . Such differences in response need not be observed in all tissues (eg, all tissues with low concentrations of adenosine-containing compounds), but only in some tissues. In certain embodiments, the side effect is an autoimmune disease (including damage to normal tissue due to excessive immune response).

In a specific aspect, the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody have substantially the same pharmaceutical effect. In certain embodiments, the pharmaceutical efficacy is substantially equal in tissues where the concentration of the adenosine-containing compound is high. Examples of tissues with high concentrations of adenosine-containing compounds include tumor tissues. Anti-CTLA-4 antibodies can be provided as pharmaceutical formulations containing them. In some embodiments, anti-CTLA-4 antibodies of the invention and control anti-CTLA-4 antibodies have substantially equal levels of immune activation in tissues with high concentrations of adenosine-containing compounds. In some embodiments, in tissues with high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the invention and the control anti-CTLA-4 antibodies require a substantially lower dose to activate immunity. exactly equal. In some embodiments, anti-CTLA-4 antibodies of the invention and control anti-CTLA-4 antibodies have substantially equal levels of therapeutic efficacy in tissues with high concentrations of adenosine-containing compounds. In some embodiments, in tissues with high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the invention and the control anti-CTLA-4 antibodies are at substantially the same dose at which a therapeutic effect is observed. be equivalent to. In certain embodiments, the therapeutic effect is the development of an anti-tumor effect (eg, tumor regression, induction of cell death or growth inhibition of tumor cells, etc.).

In certain embodiments, the tumor is selected from the group consisting of breast cancer and liver cancer.

In a further aspect, the invention provides the use of anti-CTLA-4 antibodies in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a tumor. In a further embodiment, the medicament is for use in a method of treating a tumor comprising administering an effective amount of the medicament to an individual having the tumor. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (eg, as described below). In a further embodiment, the medicament is for cell damage. In a further embodiment, the medicament is for use in a method of damaging cells in an individual, the method comprising administering to the individual an effective amount of the medicament to damage the cells. In a further embodiment, the medicament is for immune activation. In a further embodiment, the medicament is for use in a method of activating immunity in an individual, the method comprising administering to the individual an effective amount of the medicament to activate immunity. An "individual" according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method of treating a tumor. In one embodiment, the method comprises administering to an individual having such a tumor an effective amount of an anti-CTLA-4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (as described below). An "individual" according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for damaging cells in an individual. In one embodiment, the method includes administering to the individual an effective amount of an anti-CTLA-4 antibody to damage the cells. In a further aspect, the invention provides a method for activating immunity in an individual. In one embodiment, the method includes administering to the individual an effective amount of an anti-CTLA-4 antibody to activate immunity. In one embodiment, the "individual" is a human.

In a further aspect, the invention provides a pharmaceutical formulation (e.g., for use in any of the therapeutic methods described above) comprising any of the anti-CTLA-4 antibodies provided herein. . In one embodiment, the pharmaceutical formulation comprises any of the anti-CTLA-4 antibodies provided herein and a pharmaceutically acceptable carrier. In one aspect, the invention provides a pharmaceutical formulation for use in the treatment of tumors. In one aspect, the invention provides a pharmaceutical formulation for use in cell injury. In one aspect, the invention provides a pharmaceutical formulation for use in immune activation. In another embodiment, the pharmaceutical formulation comprises any of the anti-CTLA-4 antibodies provided herein and at least one additional therapeutic agent (eg, as described below).

In a further aspect, the invention provides a method for preparing a medicament or pharmaceutical formulation comprising mixing any of the anti-CTLA-4 antibodies provided herein with a pharmaceutically acceptable carrier. A method is provided (eg, for use in any of the therapeutic methods described above). In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

Antibodies of the invention can be used in therapy either alone or in combination with other agents. For example, an antibody of the invention may be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, or a chemotherapeutic agent. Examples of immune checkpoint inhibitors include anti-CTLA-4 inhibitors, anti-PD-1 inhibitors, anti-PD-L1 inhibitors, anti-PD-L2 inhibitors, anti-TIM-3 inhibitors, and anti-LAG-3 inhibitors. , anti-TIGIT inhibitors, anti-BTLA inhibitors, anti-VISTA inhibitors, etc. Examples of anti-CTLA-4 inhibitors include ipilimumab and tremelimumab. Examples of anti-PD-1 inhibitors include nivolumab and pembrolizumab. Examples of anti-PD-L1 inhibitors include atezolizumab, durvalumab, and avelumab. Examples of anti-PD-L2 inhibitors include anti-PD-L2 inhibitory antibodies. Examples of anti-TIM-3 inhibitors include anti-TIM-3 inhibitory antibodies. Examples of anti-LAG-3 inhibitors include anti-LAG-3 inhibitory antibodies. Examples of anti-TIGIT inhibitors include anti-TIGIT inhibitory antibodies. Examples of anti-BTLA inhibitors include anti-BTLA inhibitory antibodies. Examples of anti-VISTA inhibitors include anti-VISTA inhibitory antibodies. Examples of EGFR inhibitors include cetuximab, panitumumab, nimotuzumab, necitumumab, zalutumumab, and the like. Examples of HER2 inhibitors include trastuzumab, trastuzumab emtansine, pertuzumab, and the like.

Combination therapy, as described above, includes combined administration (where two or more therapeutic agents are included in the same or separate formulations), as well as separate administration, where administration of the antibodies of the invention is an additional therapy. The administration of the additional therapeutic agent may be prior to, concurrent with, and/or subsequent to the administration of the additional therapeutic agent. In one embodiment, administration of the anti-CTLA-4 antibody and administration of the additional therapeutic agent are within about 1 month, or within about 1, 2, or 3 weeks, or about 1, 2, 3, 4, 5, or within 6 days. Antibodies of the invention can also be used in combination with radiation therapy.

Antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and nasal administration, and intralesional administration if desired for local treatment. can be administered by. Parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. A variety of dosing schedules are within consideration herein, including, but not limited to, single administration or repeated administration over various time points, bolus administration, and pulse infusion.

Antibodies of the invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical presentation of the individual patient, the cause of the disorder, the site of delivery of the agent, the mode of administration, the administration schedule, and other factors known to health care professionals. The antibody is optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will typically be administered at the same doses and routes of administration as mentioned herein, or at about 1 to 99% of the doses mentioned herein, or at any dose determined to be empirically/clinically appropriate. and used in any route.

For the prevention or treatment of a disease, an appropriate dose of an antibody of the invention (when used alone or in conjunction with one or more other additional therapeutic agents) will depend on the type of disease being treated, the antibody's It will depend on the type, severity and course of the disease, whether the antibody is being administered prophylactically or therapeutically, drug history, the patient's clinical history and response to the antibody, and the discretion of the attending physician. . The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, from about 1 μg/kg to 15 mg/kg (e.g., from 0.1 mg/kg to 10 mg/kg) of antibody may be the initial candidate dose for administration to a patient. One typical daily dose may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the circumstances, treatment is usually maintained until the desired suppression of disease symptoms occurs. One exemplary dose of antibody is within the range of about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to a patient. Such doses may be administered intermittently, eg, every week or every 3 weeks (eg, such that the patient receives about 2 to about 20, or eg, about 6 doses of antibody). A high initial loading dose may be followed by one or more lower doses. The progress of this therapy is easily monitored by conventional techniques and measurements.

It will be appreciated that any of the formulations or therapeutic methods described above may be practiced using the immunoconjugates of the invention in place of or in addition to anti-CTLA-4 antibodies.

H. Articles of Manufacture In another aspect of the invention, articles of manufacture are provided that include devices useful for the treatment, prevention, and/or diagnosis of the disorders described above. The product includes a container and a label on the container or a package insert accompanying the container. Preferred containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. Containers may be formed from a variety of materials, such as glass and plastic. The container may hold the composition alone or in combination with another composition effective for treating, preventing, and/or diagnosing the condition, and may include a sterile access port. (eg, the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active ingredient in the composition is an antibody of the invention. The label or package insert indicates that the composition is to be used to treat the selected condition. Further, the article of manufacture comprises: (a) a first container with a composition comprising an antibody of the invention contained therein; and (b) a second container comprising: A second container may be included with a composition containing an additional cytotoxic or otherwise therapeutic agent contained therein. The article of manufacture in this aspect of the invention may further include a package insert indicating that the composition may be used to treat a particular condition. Alternatively or in addition, the product further comprises a second (or (a third) container. It may further include other equipment desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, and syringes.

It will be appreciated that any of the products described above may include an immunoconjugate of the invention instead of or in addition to an anti-CTLA-4 antibody.
<Polypeptide containing mutant Fc region>

In one aspect, the invention provides isolated polypeptides comprising variant Fc regions. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the variant Fc region has at least one Including amino acid residue modifications (e.g. substitutions). The native sequence Fc region is usually organized as a homodimer consisting of two identical polypeptide chains. Amino acid modifications in the mutant Fc region of the present invention may be introduced into either one of the two polypeptide chains of the parent Fc region, or into both of the two polypeptide chains.

In some aspects, the invention provides variant Fc regions that have altered function compared to a parent Fc region. In certain aspects, the variant Fc regions of the invention have enhanced binding activity for Fcγ receptors compared to the parent Fc region. In a specific embodiment, the mutant Fc region of the present invention has enhanced binding activity for at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa, as compared to the parent Fc region. In some embodiments, the mutant Fc regions of the invention have enhanced binding activity for FcγRIIa. In some embodiments, the mutant Fc regions of the invention have enhanced binding activity for FcγRIIIa. In a further embodiment, the mutant Fc region of the invention has enhanced binding activity for FcγRIIa and FcγRIIIa.

In some embodiments, the variant Fc region of the invention has at least one of at least one position selected from the group consisting of positions 234, 235, 236, 298, 330, 332, and 334, as expressed in EU numbering. Contains amino acid modifications. Alternatively, amino acid modifications described in International Publications WO2013/002362 and WO2014/104165 can be similarly used in the present invention.

In certain embodiments, the binding activity of the parent Fc region and the variant Fc region can be expressed as a KD (dissociation constant) value. In one embodiment, the ratio of [KD value of parent Fc region for FcγRIIa]/[KD value of mutant Fc region for FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In further embodiments, the FcγRIIa may be FcγRIIa R or FcγRIIa H. It may also be both. In one embodiment, the value of the ratio of [binding activity of parent Fc region to FcγRIIIa]/[binding activity of mutant Fc region to FcγRIIIa] is, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more. , 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1×10 ³ or more, 2×10 ³ or more, 3×10 ³ or more, or 5×10 ³ or more. In further embodiments, the FcγRIIIa may be FcγRIIIa F or FcγRIIIa V. It may also be both.

In one embodiment, the KD value of the mutant Fc region for FcγRIIa is, for example, 1.0×10 ⁻⁶ M or less, 5.0×10 ⁻⁷ M or less, 3.0×10 ⁻⁷ M or less, 2.0×10 ⁻⁷ M or less, 1.0× 10 ^-7 M or less, 5.0 × 10 ^-8 M or less, 3.0 × 10 ^-8 M or less, 2.0 × 10 ^-8 M or less, 1.0 × 10 ^-8 M or less, 5.0 × 10 ^-9 M or less, 3.0 × 10 ^{- 9} M or less, 2.0×10 ^-9 M or less, or 1.0×10 ^-9 M or less. In further embodiments, the FcγRIIa may be FcγRIIa R or FcγRIIa H. It may also be both. In one embodiment, the KD value of the mutant Fc region for FcγRIIIa is, for example, 1.0×10 ⁻⁶ M or less, 5.0×10 ⁻⁷ M or less, 3.0×10 ⁻⁷ M or less, 2.0×10 ⁻⁷ M or less, 1.0× 10 ^-7 M or less, 5.0 × 10 ^-8 M or less, 3.0 × 10 ^-8 M or less, 2.0 × 10 ^-8 M or less, 1.0 × 10 ^-8 M or less, 5.0 × 10 ^-9 M or less, 3.0 × 10 ^{- 9} M or less, 2.0×10 ^-9 M, 1.0×10 ^-9 M or less, 5.0×10 ^-10 M or less, 3.0×10 ^-10 M or less, 2.0×10 ^-10 M, or 1.0×10 ^-10 M or less It is. In further embodiments, the FcγRIIIa may be FcγRIIIa F or FcγRIIIa V. It may also be both.

In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed by a kd (Dissociation rate constant) value instead of a KD value.

In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as the amount of binding of the Fc region to the Fcγ receptor per unit amount. For example, in a surface plasmon resonance assay, the amount of bound Fc region immobilized on a sensor chip and the amount of Fcγ receptor bound thereto are each measured as response units (RU). The value obtained by dividing the binding amount of the Fcγ receptor by the binding amount of the Fc region can be defined as the binding amount of the Fc region to the Fcγ receptor per unit amount. Specific methods for measuring and calculating such binding amount are described in Examples below. In some embodiments, the ratio of [amount of binding of mutant Fc region to FcγRIIa]/[amount of binding of parent Fc region to FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In some embodiments, the value of the ratio of [amount of binding of mutant Fc region to FcγRIIIa]/[amount of binding of parent Fc region to FcγRIIIa] is, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more. or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1×10 ³ or more, 2×10 ³ or more, 3×10 ³ or more, or 5×10 ³ or more.

In certain embodiments, the KD value, kd value, binding amount value, etc. expressed herein are measured or calculated by performing a surface plasmon resonance assay at 25°C or 37°C (e.g., in this specification). (See Example 6 of the specification).

In certain aspects, the variant Fc regions of the invention have improved selectivity between activating and inhibitory Fcγ receptors compared to the parent Fc region. In other words, the mutant Fc region of the present invention has greater binding activity for activating Fcγ receptors than the binding activity for inhibitory Fcγ receptors, compared to the parent Fc region. In certain embodiments, the activating Fcγ receptor is at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, FcγRIIIa V, and the inhibitory Fcγ receptor is FcγRIIb. . In some embodiments, the variant Fc regions of the invention have improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the variant Fc regions of the invention have improved selectivity between FcγRIIIa and FcγRIIb. In a further embodiment, a variant Fc region of the invention has improved selectivity between FcγRIIa and FcγRIIb and between FcγRIIIa and FcγRIIb.

In some embodiments, a variant Fc region of the invention comprises at least one amino acid modification in at least one position selected from the group consisting of positions 236, 239, 268, 270, and 326, as expressed in EU numbering. . Alternatively, the amino acid modifications described in International Publications WO2013/002362 and WO2014/104165 can also be used in the present invention.

In certain embodiments, the binding activity of the parent Fc region and the variant Fc region can be expressed as a KD (dissociation constant) value. The aspects of binding activity to FcγRIIa and FcγRIIIa are as described above. In one embodiment, the ratio of [KD value of parent Fc region to FcγRIIb]/[KD value of mutant Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less. , 0.3 or less, 0.2 or less, or 0.1 or less. In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed by a kd (Dissociation rate constant) value instead of a KD value.

In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as the amount of binding of the Fc region to the Fcγ receptor per unit amount as described above. In some embodiments, the ratio of [amount of binding of mutant Fc region to FcγRIIb]/[amount of binding of parent Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5. 0.3 or less, 0.2 or less, or 0.1 or less. In some embodiments, the binding amount of the mutant Fc region to FcγRIIb is, for example, 0.5 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.005 or less, 0.003 or less, 0.002 or less, or less than 0.001.

In certain aspects, the variant Fc regions of the invention have improved stability compared to the parent Fc region. In certain embodiments, stability is thermodynamic stability. The thermodynamic stability of a polypeptide can be determined using, for example, the Tm value as an index. The Tm value can be measured using techniques known to those skilled in the art, such as CD (circular dichroism), DSC (differential scanning calorimetry), and DSF (differential scanning fluorometry). In one embodiment, the mutant Fc region of the present invention has a CH2 region Tm value of 0.1 degree or more, 0.2 degree or more, 0.3 degree or more, 0.4 degree or more, 0.5 degree or more, 1 degree or more, 2 degrees or more, compared to the parent Fc region. The temperature has increased by more than 3 degrees, 3 degrees or more, 4 degrees or more, 5 degrees or more, or 10 degrees or more.

In some embodiments, a variant Fc region of the invention comprises at least one amino acid modification in at least one position selected from the group consisting of positions 250 and 307, expressed in EU numbering. Alternatively, the amino acid modifications described in International Publication WO2013/118858 can also be used in the present invention.

In certain aspects, the variant Fc regions of the invention are composed of two polypeptide chains that differ in sequence from each other. In a further aspect, the variant Fc region of the invention has enhanced heterodimerization between the first polypeptide and the second polypeptide. When producing heterodimeric proteins using recombinant methods, different peptide chains preferentially associate with each other to form heterodimers, rather than the same polypeptide chains associating to form homodimers. It is preferable to form Whether or not heterodimerization of the mutant Fc region is promoted can be determined, for example, by separating homodimers and heterodimers from the produced mutant Fc region by a method such as chromatography, and determining the ratio of each component. It can be determined by asking for.

In some embodiments, a variant Fc region of the invention comprises at least one amino acid modification in at least one position selected from the group consisting of positions 349, 356, 366, 368, 407, and 439, as expressed in EU numbering. including. Alternatively, amino acid modifications described in International Publications WO2006/106905 and WO1996/027011 can be similarly used in the present invention.

In certain aspects, the mutant Fc region of the invention has enhanced binding activity for FcRn under acidic pH. In some embodiments, acidic pH means pH 4.0-6.5. In further embodiments, the acidic pH is pH 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 , 6.1, 6.2, 6.3, 6.4, and 6.5. In certain embodiments, the acidic pH is pH 5.8.

In some embodiments, a variant Fc region of the invention comprises at least one amino acid modification in at least one position selected from the group consisting of positions 428, 434, 436, 438, and 440, as expressed in EU numbering. . Alternatively, the amino acid modifications described in International Publication WO2016/125495 can also be used in the present invention.

In one aspect, the variant Fc region of the invention is located at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366 as expressed in EU numbering. , 368, 407, 428, 434, 436, 438, 439, and 440.

In certain aspects, variant Fc regions of the invention include amino acid alterations at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 334, as expressed in EU numbering. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 in the first polypeptide of the parent Fc region, and (ii) Contains amino acid alterations in the second polypeptide of the parent Fc region at positions 236, 250, 270, 298, 307, 326, and 334 in EU numbering.

In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 332 in EU numbering. In a further aspect, the first polypeptide of the parent Fc region comprises an amino acid modification at position 332, expressed in EU numbering.

In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 356 in EU numbering. In a further aspect, the first polypeptide of the parent Fc region comprises an amino acid modification at position 356, expressed in EU numbering.

In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 366 in EU numbering. In a further aspect, the first polypeptide of the parent Fc region comprises an amino acid modification at position 366, expressed in EU numbering.

In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 349 in EU numbering. In a further aspect, the first polypeptide of the parent Fc region comprises an amino acid modification at position 349 in EU numbering.

In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 332 in EU numbering. In a further aspect, the second polypeptide of the parent Fc region comprises an amino acid modification at position 332 in EU numbering.

In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 330 in EU numbering. In a further aspect, the second polypeptide of the parent Fc region comprises an amino acid modification at position 330 in EU numbering.

In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 439 in EU numbering. In a further aspect, the second polypeptide of the parent Fc region comprises an amino acid modification at position 439 in EU numbering.

In another aspect, the variant Fc region of the invention further comprises amino acid alterations at positions 366, 368, and 407 in EU numbering. In a further aspect, the second polypeptide of the parent Fc region comprises amino acid modifications at positions 366, 368, and 407 in EU numbering.

In another aspect, the variant Fc region of the invention further comprises an amino acid modification at position 356 in EU numbering. In a further aspect, the second polypeptide of the parent Fc region comprises an amino acid modification at position 356 in EU numbering.

In some aspects, the variant Fc region of the invention is located at position 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 349, 356, 366, 368 in EU numbering. , and an amino acid modification at 407. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 in the first polypeptide of the parent Fc region, expressed in EU numbering; and (ii) comprising amino acid modifications at positions 236, 250, 270, 298, 307, 326, 334, 356, 366, 368, and 407, expressed in EU numbering, in the second polypeptide of the parent Fc region.

In some aspects, the variant Fc regions of the invention include amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 334, 356, and 439, as expressed in EU numbering. including. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 356, expressed in EU numbering, in the first polypeptide of the parent Fc region, and (ii ) comprising amino acid modifications at positions 236, 250, 270, 298, 307, 326, 334, and 439, expressed in EU numbering, in the second polypeptide of the parent Fc region.

In some aspects, the variant Fc region of the invention is at position 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356 as expressed in EU numbering. , 366, 368, and 407. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 in the first polypeptide of the parent Fc region, expressed in EU numbering; and (ii) amino acids at positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368, and 407 in EU numbering in the second polypeptide of the parent Fc region. Including modifications.

In some aspects, the variant Fc regions of the invention are at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 356, and Contains an amino acid modification at 439. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 356 in the first polypeptide of the parent Fc region, and ( ii) comprising amino acid modifications at positions 236, 250, 270, 298, 307, 326, 330, 332, 334, and 439, expressed in EU numbering, in the second polypeptide of the parent Fc region.

In some aspects, the variant Fc region of the invention is at position 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366 in EU numbering. , 368, and 407. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349 in the first polypeptide of the parent Fc region, and 366, and (ii) amino acids at positions 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368, and 407 in EU numbering in the second polypeptide of the parent Fc region. Including modifications.

In some aspects, the variant Fc regions of the invention are at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 356, and 439 in EU numbering. Contains amino acid modifications. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 356 in the first polypeptide of the parent Fc region, expressed in EU numbering; and (ii) comprises amino acid modifications at positions 236, 250, 270, 298, 307, 326, 332, 334, and 439 in the EU numbering in the second polypeptide of the parent Fc region.

In some aspects, the variant Fc region of the invention is at position 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 366, 368 as expressed in EU numbering. , and an amino acid modification at 407. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 366 in the first polypeptide of the parent Fc region, and ( ii) comprising amino acid modifications at positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 366, 368, and 407, expressed in EU numbering, in the second polypeptide of the parent Fc region.

In some aspects, the variant Fc regions of the invention are at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 366, 368, and Contains an amino acid modification at 407. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 366 in the first polypeptide of the parent Fc region, expressed in EU numbering; and (ii) comprising amino acid modifications at positions 236, 250, 270, 298, 307, 326, 332, 334, 366, 368, and 407, expressed in EU numbering, in the second polypeptide of the parent Fc region.

In some aspects, the variant Fc region of the invention is at position 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356 as expressed in EU numbering. , 366, 368, and 407. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 349, and 366 in the first polypeptide of the parent Fc region, expressed in EU numbering; and (ii) amino acids at positions 236, 250, 270, 298, 307, 326, 330, 332, 334, 356, 366, 368, and 407 in EU numbering in the second polypeptide of the parent Fc region. Including modifications.

In some aspects, the variant Fc region of the invention is at position 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 334, 349, 356, 366 in EU numbering. , 368, and 407. In a further aspect, (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, 349 in the first polypeptide of the parent Fc region, and 366, and (ii) amino acids at positions 236, 250, 270, 298, 307, 326, 332, 334, 356, 366, 368, and 407 in EU numbering in the second polypeptide of the parent Fc region. Including modifications.

In a further embodiment, the variant Fc region of the invention comprises (i) Tyr, Phe at position 234, expressed in EU numbering; Gln at position 235; Trp at position 236; Met at position 239; Val at position 250; Asp at position 268; Glu at position 270; Ala at position 298; Pro at position 307; Asp at position 326; Glu at position 332; Cys at position 349; Lys at position 356; and Trp at position 366, and (ii) Ala at position 236 in EU numbering in the second polypeptide of the parent Fc region; Val at position 250; Glu at position 270; Ala at position 298; Pro at position 307. ; Asp at position 326; Met, Lys at position 330; Asp, Glu at position 332; Glu at position 334; Cys at position 356; Ser at position 366; Ala at position 368; Val at position 407; from Glu at position 439 At least one amino acid modification selected from the group consisting of:

In a further aspect, the variant Fc region of the invention further comprises any of the following amino acid modifications (a) to (d):
(a) Ala at position 434 in EU numbering;
(b) Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, expressed in EU numbering;
(c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, expressed in EU numbering;
(d) Leu at position 428, Ala at position 434, Arg at position 438, Glu at position 440 expressed in EU numbering.

In a further aspect, the invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 43-46, 65, 66, 81, 207, 239, 253-271, 276, 277, 278.

“Fcγ receptor” (referred to herein as Fcγ receptor, FcγR, or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and Fcγ receptor Virtually any member of a family of proteins encoded by a gene. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; -2), and FcγRII (CD32); and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and , including, but not limited to, any undiscovered human FcγR, FcγR isoform or allotype. FcγRIIb1 and FcγRIIb2 have been reported as splicing variants of human FcγRIIb. Furthermore, a splicing variant designated as FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splicing variants, human FcγRIIb includes all NCBI-registered splicing variants, NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human FcγRIIb, in addition to FcγRIIb, has all previously reported genetic polymorphisms (Arthritis Rheum. 48: 3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14: 2881- 2892 (2005); and Kyogoju et al., Arthritis Rheum. 46: 1242-1254 (2002)), and all genetic polymorphisms that will be reported in the future.

There are two allotypes of FcγRIIa, one in which the amino acid at position 131 of FcγRIIa is histidine (H type) and the other in which the amino acid at position 131 is substituted with arginine (R type) (Warrmerdam, J Exp. Med. 172: 19-25 (1990)).

FcγRs may be derived from any organism, including, but not limited to, FcγRs from humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isoform. do not have.

The amino acid sequence of human FcγRI is set forth in SEQ ID NO: 131 (NP_000557.1); the amino acid sequence of human FcγRIIa is SEQ ID NO: 132 (AAH20823.1), SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 150. The amino acid sequence of human FcγRIIb is described in SEQ ID NO: 151 (AAI46679.1), SEQ ID NO: 169, or SEQ ID NO: 172; the amino acid sequence of human FcγRIIIa is SEQ ID NO: 174 (AAH33678.1), SEQ ID NO: 175, SEQ ID NO: 176, or SEQ ID NO: 177; and the amino acid sequence of human FcγRIIIb is SEQ ID NO: 178 (AAI28563.1).

Unlike Fcγ receptors, which belong to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides and shares 22-29% sequence identity with class I MHC molecules. (Ghetie et al., Immunol. Today (1997) 18(12), 592-598). FcRn is expressed as a heterodimer consisting of a transmembrane α chain or heavy chain complexed with a soluble β chain or light chain (β2 microglobulin). Like MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, α3), with a short cytoplasmic domain anchoring the protein to the cell surface. The α1 and α2 domains interact with the FcRn-binding domain in the Fc region of antibodies (Raghavan et al. (Immunity (1994) 1, 303-315). The amino acid sequence of human FcRn is SEQ ID NO: 179 (NP_004098.1). The amino acid sequence of β2 microglobulin is set forth in SEQ ID NO: 180.

"Parent Fc region" as used herein refers to the Fc region before introduction of the amino acid modifications described herein. In some embodiments, the parent Fc region is a native sequence Fc region (or a native antibody Fc region). Antibodies include, for example, IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), and IgM. Antibodies may be derived from humans or monkeys (eg, cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, or baboons). Naturally occurring antibodies may contain naturally occurring mutations. Multiple allotype sequences of IgG due to genetic polymorphisms are described in "Sequences of proteins of immunological interest", NIH Publication No. 91-3242, any of which can be used in the present invention. In particular, for human IgG1, the amino acid sequence at positions 356-358 (EU numbering) can be either DEL or EEM. In certain embodiments, the parent Fc region is a heavy chain constant of human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), or human IgG4 (SEQ ID NO: 252). The Fc region is derived from this region. In another embodiment, the parent Fc region is an Fc region derived from the heavy chain constant region of SEQ ID NO: 82 or SEQ ID NO: 158. In a further embodiment, the parent Fc region is an Fc region created by adding amino acid modifications other than those described herein to the Fc region of the native sequence (the Fc region of the reference variant sequence); Good too. The native sequence Fc region is usually organized as a homodimer consisting of two identical polypeptide chains.

Additionally, amino acid modifications made for other purposes can be combined in the variant Fc regions described herein. For example, amino acid substitutions that enhance FcRn binding activity (Hinton et al., J. Immunol. 176(1): 346-356 (2006); Dall'Acqua et al., J. Biol. Chem. 281(33): 23514-23524 (2006); Petkova et al., Intl. Immunol. 18(12): 1759-1769 (2006); Zalevsky et al., Nat. Biotechnol. 28(2): 157-159 (2010); WO2006 /019447; WO2006/053301; and WO2009/086320), and amino acid substitutions to improve antibody heterogeneity or stability (WO2009/041613) may be added. Alternatively, a polypeptide having properties promoting antigen clearance as described in WO2011/122011, WO2012/132067, WO2013/046704, or WO2013/180201, having specific binding properties to target tissue as described in WO2013/180200 The polypeptides described in WO2009/125825, WO2012/073992, or WO2013/047752 that have the property of repeatedly binding to multiple antigen molecules can be combined with the variant Fc regions described herein. . Alternatively, the amino acid modifications disclosed in EP1752471 and EP1772465 may be combined at CH3 of the variant Fc regions described herein in order to confer binding ability to other antigens. Alternatively, amino acid modifications that reduce the pI of the constant region (WO2012/016227) may be combined in the variant Fc regions described herein for the purpose of increasing plasma retention. Alternatively, amino acid modifications that increase the pI of the constant region (WO2014/145159) may be combined in the variant Fc regions described herein for the purpose of promoting cellular uptake. Alternatively, amino acid modifications that increase the pI of the constant region (WO2016/125495) may be combined in the variant Fc regions described herein for the purpose of promoting clearance of the target molecule from plasma. In one embodiment, such modification may include a substitution at at least one position selected from the group consisting of positions 311, 343, 384, 399, 400, and 413, for example, expressed in EU numbering. . In further embodiments, such substitutions may be substitutions of amino acids at each position with Lys or Arg.

In addition, heterodimeric antibody production techniques using binding of antibodies CH1 and CL and binding of VH and VL, described in WO2011/028952, can also be used.

Similar to the methods described in WO2008/119353 and WO2011/131746, two types of homodimeric antibodies are prepared in advance, the antibodies are incubated under reducing conditions to dissociate, and then they are recombined. It is also possible to use dimeric antibody production techniques.

Furthermore, similar to the method described in WO2012/058768, it is also possible to use techniques for producing heterodimeric antibodies by modifying the CH2 and CH3 domains.

When two polypeptides containing variant Fc regions with different amino acid sequences are expressed simultaneously to generate a polypeptide containing a heterologous variant Fc region, the polypeptide containing a homologous variant Fc region as an impurity is usually also present. , is produced. In such cases, polypeptides containing heterologous variant Fc regions can be efficiently obtained by separating and purifying polypeptides containing homologous variant Fc regions using known techniques. By introducing an amino acid modification that causes a difference in isoelectric point between a homodimerized antibody and a heterodimerized antibody into the variable regions of the two types of antibody heavy chains, homodimerization is achieved using ion exchange chromatography. A method for efficiently separating and purifying heterodimeric antibodies from antibodies has been reported (WO2007/114325). To purify heterodimeric antibodies using protein A chromatography by constructing a heterodimeric antibody containing two types of heavy chains derived from mouse IgG2a, which binds to protein A, and rat IgG2b, which does not bind to protein A. Another method has been reported (WO1998/050431 and WO1995/033844).

In addition, protein A Chromatography can be used to efficiently purify heterodimeric antibodies.

In the present invention, amino acid modification means any substitution, deletion, addition, insertion, and modification, or a combination thereof. In the present invention, amino acid modification can be referred to as amino acid mutation.

The number of amino acid modifications introduced into the Fc region is not limited. In certain embodiments, it can be 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 8 or less, 10 or less, 12 or less, 14 or less, 16 or less, 18 or less, or 20 or less.

In one aspect, the invention provides a method of producing a polypeptide comprising a mutant Fc region. In a further aspect, the invention provides a method of producing a polypeptide comprising a mutant Fc region with altered function. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the methods include introducing at least one amino acid modification into the parent Fc region. In certain embodiments, the methods include (i) providing a polypeptide comprising a parent Fc region; and (ii) introducing at least one amino acid modification into the parent Fc region. In certain embodiments, the methods may further include (iii) measuring the function of a polypeptide comprising a variant Fc region. A native Fc region is usually composed of two identical polypeptide chains. Amino acid modifications to the parent Fc region may be introduced into either one of the two polypeptide chains of the parent Fc region, or into both of the two polypeptide chains.

In another embodiment, a method of producing a polypeptide comprising a variant Fc region comprises the steps of: (i) providing one or more nucleic acids encoding a polypeptide comprising a parent Fc region; (ii) converting the parent Fc region of the nucleic acid into (iii) introducing the nucleic acid produced in (ii) into a host cell, and (iv) expressing a polypeptide comprising the mutant Fc region (iii) introducing at least one mutation into the coding region; ).). In certain embodiments, the method described above may further include the step of (v) recovering a polypeptide comprising a mutant Fc region from the host cell culture described in (iv).

In certain embodiments, the nucleic acid produced in (ii) may be contained in one or more vectors (eg, expression vectors, etc.).

In some embodiments, the amino acid modification used in the present manufacturing method is any single modification, combination of single modifications, or combination of single modifications selected from among the amino acid modifications that can be included in the above-described mutant Fc region. ~Selected from the combination modifications listed in Table 30.

The Fc region may be obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc. using a protease such as pepsin, and then re-eluting the fraction adsorbed to a protein A column. The protease is not particularly limited, as long as it is capable of digesting the full-length antibody, whereby Fab and F(ab')2 are produced in a restrictive manner by appropriately setting the enzymatic reaction conditions, such as pH. , examples include pepsin and papain.

In addition to the above-described manufacturing method, the polypeptide containing the mutant Fc region of the present invention may be manufactured by other methods known in the art. Also included in the invention are polypeptides containing mutant Fc regions produced by the production methods described herein.

Any assay method or technique described herein for the purpose of identifying or screening a variant Fc region provided herein, or for determining its physical or chemical properties, or biological activity. Various measurement methods known in the art may be used.

Assays for determining the binding activity of a polypeptide comprising a variant Fc region to one or more FcR family members are described herein or known in the art. Such binding assays include, but are not limited to, surface plasmon resonance assays, amplified luminescence proximity homogeneous assay (ALPHA) screening, ELISA, and fluorescence-activated cell sorting (FACS) (Lazar et al., Proc. Natl. Acad. Sci. USA (2006) 103(11): 4005-4010).

In one embodiment, the binding activity of a polypeptide comprising a mutant Fc region to an FcR family member can be measured using a surface plasmon resonance assay. For example, immobilization or Various FcRs are allowed to interact with the captured polypeptide containing the mutant Fc region as analytes. Alternatively, FcR may be immobilized or captured on a sensor chip and polypeptides containing mutant Fc regions used as analytes. As a result of such interactions, sensorgrams of binding are obtained, which can be analyzed to calculate dissociation constant (KD) values for the binding. In addition, the difference in resonance unit (RU) values in the sensorgram before and after interaction with FcR (i.e., the amount of FcR bound) is used to determine the binding activity of the polypeptide containing the mutant Fc region to the FcR. It can also be used as an indicator. Furthermore, the difference in RU values in the sensorgrams before and after the polypeptide containing the mutant Fc region is immobilized or captured on the sensor chip (i.e., the binding amount of the polypeptide containing the mutant Fc region) A correction value obtained by dividing the amount of FcR bound (ie, the amount of FcR bound per unit amount of the polypeptide containing the mutant Fc region) may be used as an index of binding activity.

In a further aspect, the invention provides a pharmaceutical formulation comprising a polypeptide comprising a variant Fc region provided herein. In one embodiment, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier.

The following are examples of methods and compositions of the invention. It will be appreciated that various other embodiments may be implemented in light of the above general description.

[Example 0] Concept of a switch antibody that exerts antibody-dependent cytotoxic activity against cell surface markers of regulatory T cells only under cancer microenvironment Ipilimumab activates effector T cells by CTLA4 expressed on the surface of effector T cells It was thought that the antitumor effect was exerted by inhibiting CTLA4-expressing T cells, but recently it has been reported that antibody-dependent cytotoxic activity (ADCC activity) against CTLA4-expressing T cells is also important. Regulatory T cell depletion and ADCC activity have been found to be important mechanisms of action for anti-tumor effects of anti-CTLA4 antibodies.

In addition, ADCC activity by IgG1 antibodies induces cytotoxic activity when the constant region of the antibody binds to FcγR of NK cells and macrophages, and antibodies with constant regions modified to enhance this binding are more effective. It is also known to induce strong cytotoxic activity and exert antitumor effects.

On the other hand, it has been reported that removing regulatory T cells throughout the body causes a systemic reaction similar to an autoimmune disease, and the balance between the control of cytotoxic activity and systemic reaction to exert an antitumor effect is extremely important. considered to be important.

In other words, it can exert strong antitumor effects by strongly binding to regulatory T cells or exhausted T cells in the cancer microenvironment and removing regulatory T cells or exhausted T cells through cytotoxic activity. If it becomes possible to restrict it to only the environment, it is expected that it will exhibit stronger cytotoxic activity and suppress systemic reactions. No antibody with such a mechanism of action has been reported so far. Therefore, we created and verified an antibody that actually acts against CTLA4 only locally in the tumor (CTLA4 switch antibody) and has a constant region that enhances the binding of FcγR expressed on NK cells and macrophages.

[Example 1] Obtaining an antibody that binds to an antigen in the presence of ATP or its metabolites from a naive library and a rationally designed antibody library using phage display technology
(1-1) Antigen preparation for obtaining antibodies that bind to antigens in the presence of small molecules
Biotinylated mouse CTLA4 extracellular region (mCTLA4), human CTLA4 extracellular region (hCTLA4), and abatacept were prepared as antigens. Specifically, hCTLA4-His-BAP (SEQ ID NO: 1), in which a His tag and a BAP tag are fused to the C-terminus of the hCTLA4 extracellular region, was gene-synthesized and inserted into an animal expression plasmid. The antigen protein was expressed and purified using the following method. Prepared for human embryonic kidney cell-derived FreeStyle 293-F strain (Invitrogen) suspended in FreeStyle 293 Expression Medium medium (Invitrogen) at a cell density of 1.33 × 10 ⁶ cells/mL and seeded in flasks. Plasmids were introduced by lipofection. Biotin was added to a final concentration of 100 μM 3 hours after plasmid introduction, and the culture supernatant was cultured for 4 days in a CO ₂ incubator (37°C, 8% CO ₂ , 125 rpm) using a method known to those skilled in the art. The antigen was purified. The absorbance of the purified antigen solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antigen was calculated from the obtained measured value using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423). On the other hand, mCTLA4-His (Sino Biologics Inc. 50503-M08H, Accession No. NP_033973.2), which has a His tag fused to the extracellular region of mCTLA4, and abatacept (Alfresa Co., Ltd.), which has a human IgG1 constant region fused to hCTLA4, Biotinylated by amine coupling method (PIERCE Cat.No.21329).

(1-2) Obtaining antibodies that bind to mouse CTLA4 in the presence of small molecules from a naive human antibody library by bead panning. A human antibody phage display library consisting of multiple phages displaying Fab domains of different human antibody sequences was constructed according to methods known in the art.

The constructed naive human antibody phage display library was screened for antibodies whose binding activity to mouse CTLA4 extracellular region (mCTLA4) changes in the presence and absence of small molecules. That is, phages displaying antibodies that exhibit binding activity in the presence of small molecules to mCTLA4 captured on beads were collected. Phage were recovered from the phage eluate eluted from the beads in the absence of small molecules. In this acquisition method, biotin-labeled mCTLA4 (mCTLA4-His-Biotin) was used as an antigen.

The phage produced from E. coli harboring the constructed phage display phagemid was purified by a standard method. A phage library solution was then obtained by dialysis with TBS. Panning was performed using antigen immobilized on magnetic beads. NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads.

In the presence of adenosine 5'-triphosphate (ATP) and ATP metabolites, to efficiently obtain small molecule-dependent small molecule switch antibodies that can play a switch role in cancer tissues. Panning to concentrate antibodies that bind to the antigen but do not bind to the antigen in the absence of ATP was performed with reference to the method shown in prior patent WO2013/180200.

(1-3) Evaluation of binding activity in the presence and absence of small molecules by phage ELISA (1-2) From the single colony of E. coli obtained in (1-2), a conventional method (Methods Mol. Biol. (2002) 178, 133 -145), the phage-containing culture supernatant was collected. The collected culture supernatant was ultrafiltered using NucleoFast 96 (MACHERY-NAGEL). 100 μL of each culture supernatant was applied to each well of NucleoFast 96, and centrifuged at 4,500 g for 45 minutes to remove flow-through. 100 μL of H ₂ O was added, and washing was performed again by centrifugation at 4,500 g for 30 minutes. After that, 100 μL of TBS was added and the mixture was allowed to stand at room temperature for 5 minutes, and then the phage solution contained in the supernatant was collected.

The purified phages to which TBS had been added were subjected to ELISA using the following procedure. StreptaWell 96 microtiter plates (Roche) were coated overnight with 100 μL of TBS containing mCTLA4-His-Biotin. After mCTLA4-His-Biotin not bound to the plate was removed by washing each well of the plate with TBST, the wells were blocked with 250 μL of 2% skim milk-TBS for over 1 hour. After removing the 2% skim milk-TBS, the plate containing the purified phage prepared in each well was allowed to stand at room temperature for 1 hour, allowing the mCTLA4-His- It was bound to Biotin in the absence or presence of ATP. A plate to which HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS or ATP/TBS was added to each well washed with TBST or ATP/TBST was incubated for 1 hour. After washing with TBST or ATP/TBST, the color reaction of the solution in each well to which TMB single solution (ZYMED) was added was stopped by the addition of sulfuric acid, and then the color development was measured by absorbance at 450 nm. As a result, multiple antibodies were identified that bind to mCTLA4 only in the presence of ATP. The results of phage ELISA are shown in Table 2. Here, clones with an absorbance higher than 0.2 in the presence of ATP are determined to be positive, and those with a ratio of absorbance higher than 2 in the presence/absence of ATP are clones with ATP-dependent antigen binding ability (switch clones). It was determined that In this example, SM is sometimes used as an abbreviation representing a small molecule such as ATP.

(1-4) Obtaining antibodies that bind to antigens in the presence of small molecules from a rationally designed library using ATP or its metabolites. Antibodies that exhibit antigen-binding activity in the presence of metabolites (eg, ADP, AMP, adenosine (ADO), etc.) were obtained. For retrieval, phage displaying antibodies capable of binding to the antigen captured on the beads in the presence of ATP or ATP metabolites are recovered and then incubated with the beads in the absence of ATP or ATP metabolites. Phage were recovered from the eluate.

Phages were produced from E. coli harboring the constructed phage display phagemid using a standard method. A phage library solution was obtained by diluting the phage population precipitated by adding 2.5 M NaCl/10% PEG with TBS to the E. coli culture solution in which phage production had been performed. Next, BSA was added to the phage library solution to a final concentration of 4%. Panning was performed using antigen immobilized on magnetic beads. NeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (Dynabeads M-280 Streptavidin) were used as magnetic beads. Biotinylated Abatacept-Biotin was used as the antigen.

In order to efficiently obtain small molecule switch antibodies that are dependent on small molecules that can play a switch role in cancer tissues, we developed adenosine 5'-triphosphate (ATP) or ATP metabolites in the presence of adenosine 5'-triphosphate (ATP). Panning to concentrate antibodies that bind to antigen but do not bind to antigen in the absence of ATP or ATP metabolites was performed with reference to the method shown in prior patent WO2015/083764.

(1-5) Evaluation of binding activity in the presence and absence of ATP or its metabolites by phage ELISA From a single colony of E. coli obtained by the above method, a conventional method (Methods Mol. Biol. (2002) 178, 133-145), the phage-containing culture supernatant was collected. The collected culture supernatant was ultrafiltered using NucleoFast 96 (MACHEREY-NAGEL). Flow-through was removed by centrifuging (4,500 g, 45 minutes) NucleoFast 96, in which 100 μL of each culture supernatant was applied to each well. 100 μL of H ₂ O was added to each well and the NucleoFast 96 was washed again by centrifugation (4,500 g, 30 min). Finally, 100 μL of TBS was added, and the phage solution contained in the supernatant of each well of the NucleoFast 96 was collected by allowing it to stand at room temperature for 5 minutes.

Purified phage to which TBS or TBS containing ATP or its metabolites (SM/TBS) was added was subjected to ELISA using the following procedure. A StreptaWell 96 microtiter plate (Roche) was coated overnight with 100 μL of TBS containing the biotin-labeled antigen (Abatacept-Biotin) prepared in Example 1-1. After free Abatacept-Biotin was removed by washing each well of the plate with TBST, the wells were blocked with 250 μL of 2% SkimMilk-TBS for over 1 hour. After removing the 2% SkimMilk-TBS, the plate containing purified phage prepared in each well was left to stand at 37°C for 1 hour. was bound to ATP or its metabolites in the absence and presence of ATP or its metabolites. To each well washed with TBST or TBST containing ATP or its metabolites (SM/TBST), HRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) diluted in TBS or SM/TBS was added to the plate for 1 hour. Incubated. After washing with TBST or SM/TBST, the color reaction of the solution in each well to which TMB single solution (ZYMED) was added was stopped by the addition of sulfuric acid, and then the color development was measured by absorbance at 450 nm. As a result, multiple antibodies with Abatacept binding activity that changed in the presence and absence of ATP or its metabolites were identified. The results of phage ELISA are shown in Table 3. Here, clones with an absorbance S/N ratio higher than 2 in the presence of ATP or its metabolites are determined to be positive, and clones with an absorbance ratio higher than 2 in the presence/absence of ATP or its metabolites was determined to be a clone (switch clone) with antigen-binding ability dependent on ATP or its metabolites.

(1-6) Sequence analysis of a switch antibody whose antigen-binding activity changes depending on the presence or absence of ATP and its metabolites As a result of phage ELISA, it was determined that it has antigen-binding activity in the presence of ATP and its metabolites. The base sequence of the gene amplified from the clone was analyzed using specific primers lacPF (SEQ ID NO: 2) and G1seqR (SEQ ID NO: 3). As a result of analysis, clones ABADh11-4_020, ABADh11-4_086, ABADh12-4_014, ABADh12-5_001, ABADh12-5_046, and ABADh5-5_041 were obtained, which were determined to have binding activity to biotin-labeled abatacept in the presence of ATP and its metabolites. The clones were renamed as ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, respectively (Table 4).

(1-7) Expression and purification of switch antibodies whose antigen-binding activity changes depending on the presence or absence of ATP and its metabolites. The encoding gene was inserted into a human IgG1/Lambda animal expression plasmid. Antibodies were expressed using the following method. Human embryonic kidney cell-derived FreeStyle 293-F strain (Invitrogen) suspended in FreeStyle 293 Expression Medium medium (Invitrogen) at a cell density of 1.33 × 10 ⁶ cells/mL and seeded in 3 mL into each well of a 6-well plate. The prepared plasmids were introduced into the cells by lipofection. Antibodies were purified from culture supernatants cultured in a CO ₂ incubator (37° C., 8% CO ₂ , 90 rpm) for 4 days using rProtein A Sepharose™ Fast Flow (Amersham Biosciences) by a method known to those skilled in the art. The absorbance of the purified antibody solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antibody was calculated using the extinction coefficient calculated by the PACE method from the obtained measurement value (Protein Science (1995) 4, 2411-2423).

(1-8) Evaluation of the binding activity of the obtained antibodies against hCTLA4 in the presence and absence of AMP by IgG ELISA The six obtained antibodies, ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, were subjected to IgG ELISA. Ta. In addition, Buffer shown in Table 5 was appropriately prepared. Biotin-labeled human CTLA4 (hCTLA4-His-Biotin) was used as an antigen.

First, a StreptaWell 96 microtiter plate (Roche) was coated with 100 μL of TBS containing hCTLA4-His-Biotin for over 1 hour at room temperature. After washing each well of the plate with Wash buffer to remove hCTLA4-His-Biotin not bound to the plate, the wells were blocked with 250 μL of Blocking Buffer for over 1 hour. 100 μL of purified IgG adjusted to 2.5 μg/mL in Sample Buffer containing AMP at a final concentration of 1 mM was added to each well from which Blocking Buffer was removed. The plate was left to stand at room temperature for 1 hour. By doing so, each IgG was bound to hCTLA4-His-Biotin present in each well. After washing with Wash Buffer containing AMP at a final concentration of 1 mM, HRP-conjugated anti-human IgG antibody (BIOSOURCE) diluted in Sample Buffer was added to each well and the plate was incubated for 1 hour. After washing with Wash Buffer containing each low molecule, the color reaction of the solution in each well to which TMB single solution (ZYMED) was added is stopped by the addition of sulfuric acid, and the color development is measured by absorbance at 450 nm. It was done. Note that a buffer containing the composition shown in Table 5 was used as the buffer.

The measured results are shown in Table 6. For wells where the value overflowed, it was temporarily set to 5.00. In all clones ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006, the absorbance in the absence of AMP was significantly lower than that in the presence of AMP. From this result, it was confirmed that all the clones ABAM001, ABAM002, ABAM003, ABAM004, ABAM005, and ABAM006 had the property that their binding to antigens changed depending on the presence or absence of small molecules.

(1-9) Evaluation of the effects of ATP and its metabolites on binding to human CTLA4 by surface plasmon resonance
Further evaluation of ABAM004 as a CTLA4 switch antibody was performed.
The interaction of antigen-antibody reactions between ABAM004 and hCTLA4-His-BAP was analyzed using Biacore T200 (GE Healthcare). ABAM004 was captured using Sensor chip CM5 (GE Healthcare) on which an appropriate amount of protein A/G (Pierce) was immobilized using the amine coupling method, and the antigen hCTLA4-His-BAP prepared in Example 1-1 was used. made to interact. TBS was used as the running buffer, and 10 mM Glycine-HCl (pH 1.5) was used as the regeneration solution.

After 1 μg/mL ABAM004 suspended in TBS was captured, 500 nM hCTLA4-His-BAP and 10 concentrations of ATP, ADP or AMP diluted at a common ratio of 4 from 4000 μM, and ₂ mM MgCl were added. The containing solution was injected into each flow cell at a flow rate of 10 μL/min for 3 minutes. These 3 minutes were defined as the binding phase of hCTLA4-His-BAP, and the 2 minutes after the binding phase was switched to the running buffer was defined as the dissociation phase of hCTLA4-His-BAP. After the dissociation phase was completed, the regeneration solution was injected for 30 seconds at a flow rate of 30 μl/min. The above was the cycle for measuring the binding activity of ABAM004. The amount of hCTLA-4-His-BAP that interacted with ABAM004 in the binding phase was corrected for the amount of captured antibody. Biacore T200 Evaluation Software Version: 2.0 and Microsoft Excel 2013 (Microsoft) were used for data analysis and plotting.

The amount of binding between ABAM004 and hCTLA4-His-BAP in the presence of ATP and its metabolites obtained in this measurement is shown in Figure 1.

As shown in FIG. 1, ABAM004 was confirmed to have the property of binding to hCTLA4 using not only ATP but also ATP metabolites as a switch. Furthermore, it was shown that this antibody had the strongest binding activity especially in the presence of AMP.

(1-10) Evaluation of antibody binding to human CTLA4-expressing cells
Using a flow cytometer, we evaluated how the antigen-antibody interaction between ABAM004 and human CTLA4 changes in the presence and absence of AMP. CHO cells stably expressing human CTLA4 (hCTLA4-CHO cells) were prepared at appropriate concentrations. At this time, PBS containing 0.1% BSA (FACS Buffer) was used for suspension. Antibody was added to 100 μL of the cell solution at a final concentration of 10 mg/mL, then AMP was added at a final concentration of 0, 0.4, 4, 40, 200, 1000 μM, and incubated at 4°C. It was left undisturbed for 30 minutes. After that, FACS Buffer with AMP added to a final concentration of 0, 0.4, 4, 40, 200, 1000 μM was used as Wash Buffer, and after the cell line was washed, FITC-labeled secondary An antibody (Goat F(ab'2) Anti-Human IgG Mouse ads-FITC, Beckman 732598) was added, and the mixture was again left standing at 4°C in the dark for 30 minutes. After the washing operation was performed again, it was measured and analyzed using a flow cytometer (FACS CyAn ^TM ADP). The result is shown in figure 2.

The above results indicate that ABAM004 exhibits AMP concentration-dependent binding activity to hCTLA4-expressing cells, and exhibits AMP concentration-dependent binding activity not only to soluble antigens but also to membrane antigens.

(1-11) ADCC activity of test antibody using human peripheral blood mononuclear cells as effector cells
The antibody concentration-dependent ADCC activity of antibodies that bind to antigens in an ATP-dependent manner was measured according to the following method. At this time, the ADCC activity of the test antibody was measured as follows using human peripheral blood mononuclear cells (hereinafter referred to as human PBMC) as effector cells.

First, a human PBMC solution was prepared. 50 mL of peripheral blood was collected from a healthy volunteer (adult male) using a syringe in which 200 μL of 1000 units/mL heparin solution (Novo Heparin Injection 5,000 units, Novo Nordisk) was injected in advance. The peripheral blood diluted 2 times with PBS (-) was divided into 4 equal parts and placed in a Leucosep lymphocyte separation tube (Greiner bio-one) into which 15 ml of Ficoll-Paque PLUS was previously injected and centrifuged. ) was added. The separation tube into which the peripheral blood had been dispensed was centrifuged at 2150 rpm for 10 minutes at room temperature, and then the mononuclear cell fraction layer was separated. After washing the cells contained in the fraction layer once with RPMI-1640 (Nacalai Tesque) containing 10% FBS (hereinafter referred to as 10% FBS/RPMI), the cells were dissolved in 10% FBS/RPMI. The cells were suspended at a density of 1×10 ⁷ cells/mL. The cell suspension was used as a human PBMC solution in subsequent experiments.

Next, as target cells, hCTLA4-CHO cells, in which the human CTLA4 extracellular region was forced to be expressed in CHO cells, were suspended and prepared at 2×10 ⁵ cells/mL in 10% FBS/RPMI. Further, AMP (sigma) diluted to 4 mM using RPMI was used as an AMP solution for subsequent tests.

ADCC activity was evaluated by LDH (lactate dehydrogenase) release. First, 50 μL of antibody solutions prepared at various concentrations (0, 0.04, 0.4, 4, 40 μg/mL) were added to each well of a 96-well U-bottom plate, and 50 μL of target cells were seeded ( 1×10 ⁴ cells/well). Furthermore, 50 μl of AMP solution was added, and the mixture was allowed to stand at room temperature for 15 minutes. The plate in which 50 μL of human PBMC solution (5×10 ⁵ cells/well) was added to each well was centrifuged and then left standing at 37° C. for 4 hours in a 5% carbon dioxide incubator. After the reaction was completed, 100 μL of the culture supernatant was collected and transferred to a 96-well plate for measurement, and then Catalyst (C) and Dye solution (D) included in the LDH detection Kit (TaKaRa) were mixed at a ratio of 1:45. 100 μL was added. After reacting for 15 minutes at room temperature, 50 μL of 1N HCl was added to stop the reaction. Absorbance at 492 nm was measured to measure ADCC activity due to LDH release. ADCC activity was determined based on the following formula.
ADCC activity (%) = {(AD)-(CD)}×100/{(BD)-(CD)}

In the above formula, A represents the average value of LDH activity (OD492nm) in the wells to which each test antibody was added. In addition, B represents the average value of LDH activity (OD492nm) in the wells in which 10 μL of 20% Triton-X aqueous solution was added to the wells after the reaction. Furthermore, C represents the average value of LDH activity (OD492nm) in wells to which 150 μL of 10% FBS/RPMI or 100 μL of 10% FBS/RPMI and 50 μL of AMP solution were added to target cells. D represents the average value of LDH activity (OD492nm) in wells containing only 10% FBS/RPMI. The test was conducted in duplicate, and the average value of ADCC activity (%) in the test, which reflects the ADCC activity of the test antibody, was calculated. The results are shown in Figure 3.

The above results showed that the antibody ABAM004 has antigen-binding activity in the presence of AMP and has the ability to kill target cells by exerting ADCC activity.

[Example 2] Crystal structure analysis of anti-CTLA4 antibody with ATP-dependent binding properties
(2-1) X-ray crystal structure analysis of anti-CTLA4 binding antibody ABAM004 using AMP as a switch
hCTLA4-binding antibody obtained from the library in Example 1 using the AMP as a switch Fab fragment of ABAM004 alone, a complex of the Fab fragment of ABAM004 and AMP, and a complex of the Fab fragment of ABAM004, AMP, and the extracellular domain of hCTLA4. The crystal structure was analyzed.

(2-2) Preparation of ABAM004 full-length antibody for crystallization Preparation and purification of ABAM004 full-length antibody for crystallization was performed by methods known to those skilled in the art.

(2-3) Preparation of Fab fragment for crystal structure analysis of ABAM004 Fab fragment
The Fab fragment of ABAM004 was subjected to restriction digestion with rLys-C (Promega, catalog number V1671) followed by a protein A column (MabSelect SuRe, GE Healthcare) to remove the Fc fragment, and a cation exchange column (HiTrap SP HP, GE Healthcare). GE Healthcare) and loading onto a gel filtration column (Superdex200 16/60, GE Healthcare). Fractions containing Fab fragments were pooled and stored at -80°C.

(2-4) Preparation of crystals of ABAM004 Fab fragments The Fab fragments of ABAM004 for crystallization purified by the method of 2-3 were concentrated to approximately 13 mg/mL, and crystallized at 20°C by the sitting drop vapor diffusion method. Ta. The reservoir solution consisted of 0.1 M MES pH 6.5, 25% w/v polyethylene glycol 4000. The crystals thus obtained were immersed in a solution of 0.08 M MES pH 6.5, 20% w/v polyethylene glycol 4000, 20% ethylene glycol.

(2-5) Collection of X-ray diffraction data and structure determination from crystals of ABAM004 Fab fragments
X-ray diffraction data were measured with BL-17A at the Photon Factory, a synchrotron radiation facility of the High Energy Accelerator Research Organization. During the measurements, the crystals were constantly placed under a nitrogen flow at -178°C to maintain a frozen state, and the crystals were rotated 0.5° at a time using a Quantum 270 CCD detector (ADSC) connected to the beamline. A total of 360 X-ray diffraction images were collected. Determination of cell parameters, indexing of diffraction spots, and processing of diffraction data obtained from diffraction images were performed using the Xia2 program (J. Appl. Cryst. 43: 186-190 (2010)) and the XDS package (Acta. Cryst. D66: 125-132 (2010)) and Scala (Acta. Cryst. D62: 72-82 (2006)), and finally obtained diffraction intensity data with a resolution of 1.70 Å. Crystallographic data statistics are shown in Table 7.

The structure was determined by molecular replacement using the program Phaser (J. Appl. Cryst. (2007) 40, 658-674). The search model for Fab fragments was derived from the published crystal structure of Fab (PDB code: 4NKI). The model was built with the Coot program (Acta Cryst. D66: 486-501 (2010)) and refined with the program Refmac5 (Acta Cryst. D67: 355-467 (2011)). The crystallographic reliability factor (R) of the diffraction intensity data from 52.92-1.70 Å was 16.92%, and the Free R value was 21.22%. Structure refinement statistics are shown in Table 7.

(2-6) ABAM004 Fab fragment and AMP complex, and preparation of ABAM004 Fab fragment from full-length antibody for crystal structure analysis of AMP and hCTLA4 complex
The Fab fragment of ABAM004 was subjected to restriction digestion with papain (Roche Diagnostics, catalog number 1047825) followed by a protein A column (MabSelect SuRe, GE Healthcare) to remove the Fc fragment, and a cation exchange column (HiTrap SP HP, GE Healthcare). ) and loading onto a gel filtration column (Superdex200 16/60, GE Healthcare). Fractions containing Fab fragments were pooled and stored at -80°C.

(2-7) Preparation of crystals of ABAM004 Fab fragment and AMP complex The Fab fragment of ABAM004 for crystallization purified by the method 2-6 was concentrated to approximately 13 mg/mL, and the final concentration was 2 mM. AMP was added so that The reservoir solution consisted of 0.1 M Morpheus buffer 2 pH 7.5, 37.5% w/v MPD_P1K_P3350, 10% Morpheus Carboxylic acids (Morpheus, Molecular Dimensions).

(2-8) Collection of X-ray diffraction data and structure determination from crystals of ABAM004 Fab fragment and AMP complex
X-ray diffraction data were measured with BL-1A at the Photon Factory, a synchrotron radiation facility at the High Energy Accelerator Research Organization. During the measurements, the crystal was kept frozen under a nitrogen flow at -178°C, and the crystal was rotated 0.25° at a time using a Pilatus 2M detector (DECTRIS) connected to the beamline, and the total 720 X-ray diffraction images were collected. Determination of cell parameters, indexing of diffraction spots, and processing of diffraction data obtained from diffraction images were performed using the Xia2 program (J. Appl. Cryst. 43: 186-190 (2010)) and the XDS package (Acta. Cryst. D66: 125-132 (2010)) and Scala (Acta. Cryst. D62: 72-82 (2006)), and finally obtained diffraction intensity data with a resolution of 1.70 Å. Crystallographic data statistics are shown in Table 7.

The structure was determined by molecular replacement using the program Phaser (J. Appl. Cryst. (2007) 40, 658-674). The search model for Fab fragments was derived from the published crystal structure of Fab (PDB code: 4NKI). The model was built with the Coot program (Acta Cryst. D66: 486-501 (2010)) and refined with the program Refmac5 (Acta Cryst. D67: 355-367 (2011)). The crystallographic reliability factor (R) of the diffraction intensity data from 47.33-2.89 Å was 19.97%, and the Free R value was 25.62%. Structure refinement statistics are shown in Table 7.

(2-9) Preparation of hCTLA4 extracellular domain
The extracellular domain of hCTLA4 was isolated using abatacept, restriction digestion with Endoproteinase Lys-C (Roche, catalog number 11047825001), followed by a protein A column (MabSelect SuRe, GE Healthcare) to remove Fc fragments, and a gel filtration column (MabSelect SuRe, GE Healthcare). It was prepared by loading onto Superdex200 10/300 (GE Healthcare). Fractions containing the extracellular domain of hCTLA4 were pooled and stored at -80°C.

(2-10) Preparation of complex of ABAM004 Fab fragment, AMP, and hCTLA4 extracellular domain. They were mixed at a molar ratio and AMP was added to a final concentration of 2 mM. The complex was purified by gel filtration chromatography (Superdex200 10/300, GE Healthcare) using a column equilibrated with 25mM HEPES pH7.5, 100mM NaCl, 2mM AMP.

(2-11) Preparation of crystals of ABAM004 Fab fragment, AMP, and hCTLA4 extracellular domain complex The purified complex was concentrated to about 8 mg/mL and incubated at 20°C by the sitting drop vapor diffusion method combined with the seeding method. Crystallization was performed. The reservoir solution consisted of 0.1 M Morpheus buffer 1 pH 6.5, 37.5% w/v M1K3350, 10% halogens (Morpheus, Molecular Dimensions).

(2-12) Collection of X-ray diffraction data and structure determination from crystals of ABAM004 Fab fragment, AMP and hCTLA4 extracellular domain complex
X-ray diffraction data were measured with BL32XU at SPring-8. During the measurement, the crystal was constantly placed under a nitrogen flow at -178°C to maintain a frozen state, and the crystal was rotated 1.0° at a time using an MX-225HS CCD detector (RAYONIX) connected to the beamline. , a total of 180 X-ray diffraction images were collected. Determination of cell parameters, indexing of diffraction spots, and processing of diffraction data obtained from diffraction images were performed using the Xia2 program (J. Appl. Cryst. 43: 186-190 (2010)) and the XDS package (Acta. Cryst. D66: 125-132 (2010)) and Scala (Acta. Cryst. D62: 72-82 (2006)), and finally obtained diffraction intensity data with a resolution of 1.70 Å. Crystallographic data statistics are shown in Table 7.

The structure was determined by molecular replacement using the program Phaser (J. Appl. Cryst. (2007) 40, 658-674). The search model for Fab fragments was derived from the published crystal structure of Fab (PDB code: 4NKI), and the search model for the extracellular domain of hCTLA4 was derived from the published crystal structure of human CTLA4 (PDB code: 3OSK, J. Biol. Chem. 286: 6685-6696 (2011)). The model was built with the Coot program (Acta Cryst. D66: 486-501 (2010)) and refined with the program Refmac5 (Acta Cryst. D67: 355-367 (2011)). The crystallographic reliability factor (R) of the diffraction intensity data from 52.81-3.09 Å was 23.49%, and the Free R value was 31.02%. Structure refinement statistics are shown in Table 7.

(2-13) Identification of the interaction site between ABAM004 and AMP From this crystal structure, it was found that AMP is mainly recognized by the heavy chain of the antibody.
The adenine ring portion of AMP is recognized by heavy chain CDRs 1 and 3, and the ribose portion and phosphate group portion are recognized by CDRs 1 and 2.

Specifically, as shown in Figure 4, the adenine ring portion of AMP includes the T33 belonging to the heavy chain CDR1 of the antibody, the Y95, L100B, and W100C side chains belonging to CDR3, and the main chains G96 and M100A. recognized by In particular, the main chain carbonyl oxygen of G96 and M100A forms a hydrogen bond with N at position 6 of AMP, and the main chain amide NH group of W100C forms a hydrogen bond with N at position 1 of Y95, L100B, and W100C. It was found that the antibody strongly recognizes the adenine ring portion by forming an interaction between the side chain and the pi electrons possessed by the adenine ring portion. The ribose moiety is recognized by each side chain of T33 belonging to heavy chain CDR1 and Y56 and Y58 belonging to CDR2 through van der Waals interaction and interaction with pi electrons possessed by Y56. Furthermore, the phosphate group moiety is recognized by the side chains of T33 belonging to heavy chain CDR1, S52, S52A, and R53 belonging to CDR2, and the main chain of S52A. In particular, the hydrogen bond formed between the side chain of T33 and the main chain amide NH group of S52A with the phosphate group, and the van der Waals interaction between S52 and R53 play an important role in the recognition of the phosphate group. It is thought that there are. Note that the amino acid residue numbering of Fab is based on the Kabat numbering scheme.

(2-14) Identification of epitope of ABAM004 In Figures 5 and 6, the epitope of ABAM004 Fab contact region is mapped in the hCTLA4 crystal structure and amino acid sequence, respectively. The epitope includes amino acid residues of hCTLA4 that contain one or more non-hydrogen atoms that are located within 4.2 Å from any portion of ABAM004 Fab or AMP in the crystal structure.
At least antigens M3, E33, R35, T53, E97, M99, Y100, P101, P102, P103, Y104, Y105, and L106 are recognized by heavy chain CDR2, CDR3, light chain CDR1, CDR3, and AMP of the antibody. The crystal structure revealed that In particular, the loop consisting of M99 to Y104 of the antigen is strongly recognized by the antibody so that it is buried within the CDR loop of the antibody, and is thought to play a major role in antigen recognition by the antibody.

(2-15) Mechanism of AMP-dependent antigen binding Figure 7 shows the crystal structure of ABAM004 Fab alone, the complex of ABAM004 Fab and AMP, and the crystal structure of the ternary complex consisting of ABAM004 Fab, AMP, and CTLA4. It is a diagram in which the variable regions of the antibody are extracted from the structure and superimposed with the heavy chain in the center. For AMP-dependent antigen binding, it is thought that not only the direct interaction between AMP and CTLA4 shown in Example 2-14 but also the structural change of the antibody that occurs upon binding of AMP is important.

As shown in Figure 7, by comparing the single crystal structure of the antibody and the crystal structure in which AMP is bound to the antibody, it is possible to determine the loop structure of heavy chain CDR3 and light chain CDR3, as well as the structure of the antibody in the variable region. It was revealed that the torsion angle between the heavy and light chains was also changed. Furthermore, when comparing the crystal structure of the antibody bound to AMP and the crystal structure of the ternary complex consisting of the antibody, AMP, and CTLA4, it was found that the structure of heavy chain CDR3 and light chain CDR1 further changes upon antigen binding. Antigen-dependent structural changes are also observed. On the other hand, since there is no change in the loop structure of light chain CDR3 and the torsion angle between the heavy chain and light chain, binding of AMP changes the structure of the antibody to a state close to the structure upon antigen binding. It is thought that this may have been the case. Therefore, the structural changes associated with AMP binding are necessary to form an appropriate structure for antigen binding, and are thought to play an important role in AMP-dependent antigen binding.

[Example 3] Production of modified CTLA4 antibody and evaluation of its activity
(3-1) Preparation of CTLA4-binding activity enhanced variant of ABAM004 antibody
ABAM004 (VH SEQ ID NO: 10, VL SEQ ID NO: 11) obtained from the human Rational Design phage library described in Example 1 has a modified amino acid sequence, and the CTLA4 binding activity in the absence of the ATP analog is different from that sequence. human CTLA4 binding activity in the presence of ATP analogs was decreased and binding to ATP and ATP analogs was improved. For this purpose, point mutations of residues predicted to be involved in the binding based on the co-crystal structure of ABAM004 and AMP obtained by the method described in Example 2 and the co-crystal structure of ABAM004, AMP, and human CTLA4 body was created. Furthermore, variants were created in which all amino acids in the CDR were replaced with Ala or Pro. The human CTLA4 (Abatacept and hCTLA4-His-BAP) binding activity of this point mutant was measured in the absence of ATP and in the presence of ATP, ADP, or AMP using Biacore T200 or Biacore 4000 (GE Healthcare), and the binding activity was improved. Mutations that cause this were screened. Mutants were created by combining mutations that improved binding activity, and KD values were calculated using Biacore. As a result, it was revealed that replacing the heavy chain of ABAM004 with H32A and S52aT and the light chain with T24D, T26P, and E50F (numbers are Kabat numbers) improved the binding properties of ABAM004. The mutant is designated 04H0150/04L0072 (VH SEQ ID NO: 47, VL SEQ ID NO: 48).

(3-2) Measurement of the influence of ATP and its metabolites on the human CTLA4 binding activity of ABAM004 and 04H0150/04L0072 by surface plasmon resonance First, a chip was created for Biacore T200 measurement. The temperature setting of Biacore T200 was 25°C, and the flow rate was 10 μL/min. HBS-EP+ was used as the running buffer. An equal volume mixture of NHS (N-hydroxysuccinimide) and EDC (N-ethyl-N'-(dimethylaminopropyl) carbodiimide) was added to Sensor chip CM5 (GE Healthcare) at a flow rate of 10 μL/min for 10 minutes, and the flow cell was activated. Next, 25 μg/mL Protein A/G (Pierce) suspended in 10 mM sodium acetate pH 4.0 was added at 10 μL/min for 30 minutes for binding. Thereafter, excess active groups on the flow cell were blocked by adding 1 M ethanolamine-HCl at 10 μL/min for 10 minutes.

Next, the influence of ATP and its metabolites on the binding of the target antibody to human CTLA4 was measured. The set temperature was 25°C, and TBS was used as the running buffer. 10 mM Glycine-HCl (pH 1.5) was used as the regeneration solution. After the antibodies suspended in TBS were captured, a TBS solution containing 500 nM hCTLA4-His-BAP, 10 concentrations of ATP, ADP or AMP diluted at a common ratio of 4 from 4000 μM, and ₂ mM MgCl was added to each sample. It was injected into a flow cell for 3 minutes at a flow rate of 10 μL/min. This 3 minutes was used as the binding phase of hCTLA4-His-BAP. The dissociation phase was defined as the 2-minute period after the binding phase and the switching to the running buffer. After the dissociation phase was completed, the regeneration solution was injected for 30 seconds at a flow rate of 30 μL/min, and the above was considered as a binding activity measurement cycle. The binding amount of hCTLA4-His-BAP that interacted with ABAM004 and 04H0150/04L0072 in the binding phase was corrected by the amount of captured antibody and is shown in FIG. In addition, in the binding measurements of ABAM004 and 04H0150/04L0072, the small molecule concentration in the bonded phase was kept at 62.5 μM or 1 mM, and 8 concentrations of hCTLA4-His-BAP diluted with a common ratio of 2 from 2000 nM were used in the bonded phase. Ta. The KD values obtained from the analysis of the binding amount of hCTLA4-His-BAP are shown in Table 8. Biacore T200 Evaluation Software Version:2.0 and Microsoft Excel 2013 (Microsoft) were used for data analysis and plotting. A steady state affinity model was used to calculate the KD value.

SMは、それぞれの測定で用いられた小分子（ATP、ADPまたはAMP）を表す。

SM represents the small molecule (ATP, ADP or AMP) used in the respective measurement.

(3-3) In order to create an anti-CTLA4 antibody with superior binding ability due to the introduction of comprehensive modifications , we used 04H0150/04L0072, which is the variable region of the anti-human CTLA4 antibody created in Example 3-1. Amino acid modifications were comprehensively introduced. Substitutions for all 18 amino acids excluding cysteine were created for all CDRs of 04H0150 and 04L0072 by methods known to those skilled in the art such as PCR. The binding of the approximately 1200 produced variants to human CTLA4 was measured using Biacore 4000. Antibodies were captured by interacting culture supernatants of antibody variants with a series S Sensor Chip CM5 (GE Healthcare) immobilized with Protein A/G (Thermo Fisher Scientific). Next, human CTLA4 solutions with or without small molecules (ATP, ADP, or AMP) were allowed to interact with antibodies in the presence and absence of small molecules. The ability to bind to human CTLA4 was evaluated. Measurements were performed at 25°C using Tris buffered saline, 0.02% PS20 as a running buffer.

Modifications found using the above method that enhance binding to human CTLA4 in the presence of small molecules and modifications that reduce binding to human CTLA4 in the absence of small molecules are combined to provide better An anti-human CTLA4 antibody showing the profile was generated. Gene of antibody heavy chain 04H0150-G1m (SEQ ID NO: 209), which has 04H0150 as the heavy chain variable region and G1m (SEQ ID NO: 82) with Gly and Lys removed from the C-terminus of human IgG1 as the heavy chain constant region An antibody heavy chain gene was created by combining the modifications discovered through comprehensive modification introduction and the modifications to the Framework. In addition, the modification found in the antibody light chain 04L0072-lam1 (SEQ ID NO: 208), which has 04L0072 as a light chain variable region and human λ chain lam1 (SEQ ID NO: 87) as a light chain constant region, was combined. An antibody light chain gene was created. Furthermore, a variant in which the framework and constant region of the light chain variable region were replaced with the human κ chain sequence was also created. In addition, for comparison, the gene of the antibody heavy chain MDX10D1H-G1m (SEQ ID NO: 210) having the heavy chain variable region MDX10D1H (SEQ ID NO: 154) of the existing anti-human CTLA4 antibody described in WO0114424 and the light chain variable region MDX10D1L (SEQ ID NO: 210) were used for comparison. A gene for antibody light chain MDX10D1L-k0MT (SEQ ID NO: 211) having SEQ ID NO: 155) was created. An antibody was expressed and purified using a combination of these genes by a method known to those skilled in the art, and the desired anti-CTLA4 antibody was produced. Table 9 lists the sequence numbers of the heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, and hypervariable region of the produced antibodies.
The antibodies herein are named according to the following rules: (heavy chain variable region)-(heavy chain constant region)/(light chain variable region)-(light chain constant region). For example, if the antibody name is 04H0150-G1m/04L0072-lam1, the heavy chain variable region of this antibody is 04H0150, the heavy chain constant region is G1m, the light chain variable region is 04L0072, and the light chain constant region is lam1. means.

Amino acid sequences of heavy chains, light chains, and their hypervariable regions (indicated by SEQ ID NOs)

Binding of the produced antibody to human CTLA4 was measured using Biacore T200. The running buffer used was 20mM ACES (pH 7.4), 150mM NaCl, 2mM _MgCl2 , 0.05% Tween20 with ATP added to the desired concentration, and measurements were performed at 37°C. . First, antibodies were captured by interacting an antibody solution prepared with an ATP-free running buffer with a Series S Sensor Chip CM3 (GE Healthcare) immobilized with Protein G (CALBIOCHEM). Next, by interacting a human CTLA4 solution prepared in a running buffer with ATP added or a human CTLA4 solution prepared in a running buffer without ATP to the desired concentration, in the presence of ATP and The binding ability of the antibody to human CTLA4 in the absence of ATP was evaluated. The chip was regenerated with 25 mM NaOH and 10 mM Glycine-HCl (pH 1.5), and antibodies were captured and measured repeatedly. The dissociation constant of each antibody for CTLA4 was calculated using Biacore T200 Evaluation Software 2.0. Specifically, the binding rate constant ka (L/mol/s) and dissociation rate constant kd (1/s) are calculated by global fitting the sensorgram obtained from the measurement using a 1:1 Langmuir binding model. The dissociation constant KD (mol/L) was calculated from that value. Alternatively, the dissociation constant KD (mol/L) was calculated using a steady state model. In addition, the amount of CTLA4 bound per unit amount of antibody was calculated by correcting the amount of bound CTLA4 determined from the sensorgram obtained by the measurement with the amount of antibody captured on the chip surface. Table 10 shows the results of these measurements.

Binding analysis of modified antibodies to human CTLA4

The value of "binding to human CTLA4" in the table indicates the amount of human CTLA4 bound per unit amount of antibody when interacting with human CTLA4 at 1000 nM under each ATP concentration condition described. "KD (M)" indicates the dissociation constant for human CTLA4 under each ATP concentration condition. KD values marked with * in the table were calculated using a steady state model. All variants produced using 04H0150-G1m/04L0072-lam1 as a parent antibody were shown to have enhanced binding in the presence of ATP compared to 04H0150-G1m/04L0072-lam1. In addition, 04H0150-G1m/04L0072-lam1 and these variants had a higher binding amount in the presence of 10 μM than in the presence of 1 μM ATP, and also had a higher binding amount in the presence of 100 μM. It was shown to bind to human CTLA4 in an ATP concentration-dependent manner. On the other hand, the comparison target MDX10D1H-G1m/MDX10D1L-k0MT did not show such ATP concentration-dependent binding to human CTLA4. 04H1077-G1m/04L1305-k0MT, which has the light chain Framework and constant region of 04H1077-G1m/04L1086-lam1 replaced with human κ chain, has higher binding to human CTLA4 in the absence of ATP than 04H1077-G1m/04L1086-lam1. was enhanced, but ATP concentration-dependent binding was also enhanced. These results showed that even after substitution with the human κ chain sequence, the property of binding to human CTLA4 in an ATP-dependent manner was maintained. Among the antibodies produced here, 04H1077-G1m/04L1066-lam1, 04H1077-G1m/04L1305-k0MT, and 04H1207-G1m/04L1086-lam1 are existing anti-human CTLA4 antibodies in the presence of 100 μM ATP. It showed almost the same binding activity as MDX10D1H-G1m/MDX10D1L-k0MT, and 04H1208-G1m/04L1407-k0MT showed stronger binding activity than MDX10D1H-G1m/MDX10D1L-k0MT in the presence of 10 μM or more of ATP.

Next, among the antibodies produced in Table 10, the binding of 04H1077-G1m/04L1086-lam1 and 04H1208-G1m/04L1407-k0MT to mouse CTLA4 was evaluated. For comparison, the gene of the antibody heavy chain hUH02-G1d (SEQ ID NO: 212) having the heavy chain variable region hUH02 (SEQ ID NO: 156) of anti-mouse CTLA4 antibody and the gene having the light chain variable region hUL01 (SEQ ID NO: 157) The gene for antibody light chain hUL01-k0 (SEQ ID NO: 213) was created, expressed, purified, and used. Measurements were performed using mouse CTLA4 as a sample and Biacore T200 under the same conditions as in the binding measurement to human CTLA4 (Table 11). Mouse CTLA4 was prepared as follows.
A gene in which a His tag was linked to the extracellular region of mouse CTLA4 (mCTLA4-His) (SEQ ID NO: 49) was synthesized and inserted into an animal expression plasmid. Prepared for human embryonic kidney cell-derived FreeStyle 293-F strain (Invitrogen) suspended in FreeStyle 293 Expression Medium medium (Invitrogen) at a cell density of 1.33 × 10 ⁶ cells/mL and seeded in flasks. Plasmids were introduced by lipofection. The absorbance of the purified antigen solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antigen was calculated from the obtained measured value using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

Binding analysis of modified antibodies to mouse CTLA4

The value of "binding to mouse CTLA4" in the table indicates the amount of mouse CTLA4 bound per unit amount of antibody when interacting with mouse CTLA4 at 1000 nM under each ATP concentration condition listed. KD (M) for CTLA4 indicates the dissociation constant for mouse CTLA4 under each ATP concentration condition. hUH02-G1d/hUL01-k0 binds to mouse CTLA4 to the same extent regardless of ATP concentration, whereas 04H1077-G1m-04L1086-lam1 and 04H1208-G1m/04L1407-k0MT both bind to mouse CTLA4 in an ATP concentration-dependent manner. was shown to bind to mouse CTLA4. Compared to the binding ability to human CTLA4 shown in Table 10, in the presence of 100 μM ATP, the binding ability of 4H1077-G1m-04L1086-lam1 to mouse CTLA4 is approximately 5% compared to the binding ability to human CTLA4. The ability of 04H1208-G1m/04L1407-k0MT to bind to mouse CTLA4 was about twice as weak as that to human CTLA4.

(3-4) Preparation of anti-mCTLA4 control antibody and anti-mCTLA4 switch antibody Anti-mCTLA4 control antibody (hUH02-mFa55/hUL01-mk1 abbreviation: mNS-mFa55) and anti-CTLA4 switch (04H1077-mFa55/04L1086-ml0r abbreviation: SW1077-mFa55, 04H1208-mFa55/04L1407s-mk1 (abbreviation: SW1208-mFa55) were produced. The mNS-mFa55 antibody uses the heavy chain variable region hUH02 (SEQ ID NO: 16) and the light chain variable region hUL01 (SEQ ID NO: 17), and the constant regions are the mouse heavy chain constant region mFa55 (SEQ ID NO: 18) and the wild type Mouse light chain constant region mk1 (SEQ ID NO: 19) was used. At this time, a mouse heavy chain constant region modified to enhance binding to Fcγ receptors was used and expressed and purified by a method known to those skilled in the art.
SW1077-mFa55 antibody uses heavy chain variable region 04H1077 (SEQ ID NO: 20) and light chain variable region 04L1086 (SEQ ID NO: 21), and the constant region is mouse heavy chain constant region mFa55 (SEQ ID NO: 18) and wild type Mouse light chain constant region ml0r (SEQ ID NO: 22) was used. At this time, a mouse heavy chain constant region modified to enhance binding to Fcγ receptors was used and expressed and purified by a method known to those skilled in the art.
SW1208-mFa55 antibody uses heavy chain variable region 04H1208 (SEQ ID NO: 23) and light chain variable region 04L1407s (SEQ ID NO: 24), and the constant region is mouse heavy chain constant region mFa55 (SEQ ID NO: 18) and wild type Mouse light chain constant region mk1 (SEQ ID NO: 19) was used. At this time, a mouse heavy chain constant region modified to enhance binding to Fcγ receptors was used and expressed and purified by a method known to those skilled in the art.

(3-6) Evaluation of neutralizing activity of CTLA4 switch antibody The neutralizing activity of the anti-CTLA4 switch antibody (SW1077-mFa55 prepared in Example 3-4) was evaluated by competitive ELISA. In a 96-well plate, mCTLA4-Fc (SEQ ID NO: 25), which is mCTLA4 linked to a human constant region, was diluted to 5 μg/mL (55 nM) with 0.1 M NaHCO ₃ , 0.05% NaN _{3 .} -Fc solution was added in 100 μL portions and allowed to stand overnight at 4°C to immobilize it on the plate surface. After washing three times with TBS and 0.1% Tween20, 250 μL of a BSA solution diluted to 2% with TBS was added to each well to block the plate surface. After that, I washed it 3 times. mCD86-Fc-His, human constant region and His tag fused to mouse CD86 (Sino Biologics Inc. 50068-M03H, Accession No. NP_062261.3), diluted in TBS to a final concentration of 55 nM, final concentration SW1077-mFa55 antibody solution diluted to give a final concentration of 6.25, 1.56, 0.390, 0.0977, 0.0061, 0 μg/mL, and ATP solution diluted to a final concentration of 0, 1, 10, 100 μM, respectively. The mixture was mixed to a total of 100 μL, added to each well, and left at 37° C. for 1 hour. Thereafter, each well was washed three times with TBS, 0.1% Tween20, which was prepared to contain the same ATP concentration as the ATP concentration of the solution added to each well. 100 μL of anti-His-tag mAb-HRP-Direct (MBL Life Sciences) diluted 10,000 times with blocking buffer to contain the same ATP concentration as the solution added to each well was added to each well for 1 hour. It was left standing at 37°C. Thereafter, each well was washed three times with TBS, 0.1% Tween20, which was prepared to contain the same ATP concentration as the ATP concentration of the solution added to each well. Then, add 100 μL of TMB solution to each well, leave it at 37°C for 1 hour, stop the reaction by adding 50 μL of 1M H ₂ SO ₄ to each well, and measure the absorbance at 450 nm with an absorbance microplate reader (Wako Sunrise). was detected.
The absorbance value of the wells to which no antibody was added under the same ATP concentration conditions was defined as the mCTLA4-mCD86 binding rate of 100%, and the extent to which the binding rate decreased with the addition of the antibody was evaluated. The results are shown in Figure 9.

This result showed that the neutralizing activity of the SW1077 antibody against the mCTLA4-mCD86 interaction became stronger as the ATP concentration in the assay increased. This result confirmed that the SW1077 antibody has ATP-dependent neutralizing activity.

(3-7) Efficacy of anti-CTLA4 switch antibody in a mouse model transplanted with syngeneic tumor cells, increase and decrease of regulatory T (Treg) cells in the tumor, and movement of systemic response markers in the spleen
(3-7-1) Preparation of mouse model with cell line and syngeneic tumor line transplantation Mouse breast cancer line FM3A cells purchased from RIKEN were used as cells. FM3A cells were maintained and subcultured in RPMI1640 medium (SIGMA) containing 10% Bovine serum (Thermo Fisher Scientific). The mice used were C3H/HeN mice (7 weeks old, male) purchased from Charles River Japan. FM3A cells were transplanted subcutaneously into the abdomen of mice, and the model was established when the volume of the transplanted tumor increased from approximately 150 mm ³ to 300 mm ³ .

The volume of the transplanted tumor was calculated using the following formula.
Tumor volume = long axis x short axis x short axis/2

(3-7-2) Preparation of drug to be administered
The drugs administered to the FM3A cell transplant model were the anti-mouse CTLA4 control antibody (mNS-mFa55) and anti-CTLA4 switch antibody (SW1208-mFa55) prepared in Example 3-4. mNS-mFa55 is 0.0005 mg/mL, 0.005 mg/mL, 0.0125 mg/mL, 0.05 mg/mL, 0.5 mg/mL, 1.5 mg/mL, 5 mg/mL, SW1208-mFa55 is 0.005 mg/mL , 0.05 mg/mL, 0.5 mg/mL, 5 mg/mL, and 25 mg/mL using His-buffer (20 mM His-HCl, 150 mM NaCl, pH 6.0), respectively.

(3-7-3) Drug administration for antitumor effect measurement On the 9th day after transplantation, mNS-mFa55 was administered at 0.01 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 1 mg/kg, 10 mg/kg. , SW1208-mFa55 was administered to mice at doses of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and 500 mg/kg, respectively. did. Administration was performed through the tail vein at a dose of 20 mL/kg of the prepared administration solution.
Table 12 shows details regarding drug treatment when measuring antitumor effects.

Measurement of antitumor effect in FM3A cell transplantation model

(3-7-4) Evaluation of anti-tumor effect The anti-tumor effect was evaluated using the tumor volume calculated using the formula described in (3-7-1).

The tumor growth inhibition rate (TGI) value was calculated using the following formula.
TGI (%) = (1 - (average tumor volume of the group of interest at the time of measurement - average value of tumor volume of the group of interest at the time of first administration) ÷ (average of tumor volume of the control group at the time of measurement - Average tumor volume in control group at first administration))×100

As a result, mNS-mFa55 at a dose of 0.1 mg/kg or more and SW1208-mFa55 at a dose of 1 mg/kg or more showed efficacy of TGI = 60% or more on the 13th day after administration (Figures 10 and 11 ).

(3-7-5) Drug administration for evaluation of intratumoral Treg cells and verification of systemic effects in the spleen 7 days after transplantation, anti-mouse CTLA4 control antibody (mNS-mFa55) at 0.1 mg/kg, 1 mg Anti-CTLA4 switch antibody (SW1208-mFa55) was administered via the tail vein at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, It was administered via the tail vein at 500 mg/kg. Details regarding drug treatment during evaluation of intratumoral Treg cells and verification of systemic effects in the spleen are shown in Table 13.

Verification of intratumoral and systemic effects in FM3A cell transplantation model (mNS-mFa55 and SW1208-mFa55)

(3-7-6) Removal of tumor and spleen from FM3A cell transplanted model mouse Six days after administration of the antibody, the mouse was euthanized under anesthesia, and the tumor and spleen were removed. Prepare a cell suspension from the removed spleen using RPMI-1640 medium (SIGMA) containing 10% FBS (SIGMA), then perform hemolysis using the Mouse Erythrocyte Lysing kit (R&D) to prepare spleen cells. did. The removed tumor was crushed using a Tumor dissociation kit, mouse (miltenyi). Both spleen cells and crushed tumors were reacted with the following antibodies, and the fraction of immune cells present was analyzed by FACS analysis. Anti-CD45 antibody (BD, clone: 30-F11), anti-CD3 antibody (BD, clone: 145-2C11), anti-CD4 antibody (BD, clone: RM4-5), anti-FoxP3 antibody (eBioscience, clone: FJK-16s) ), anti-ICOS antibody (eBioscience, clone: 7E17G9), anti-KLRG1 antibody (Biolegend, clone: 2F1/KLRG1). FACS analysis was performed on a BD LSRFortessa X-20 (BD).

(3-7-7) Tumor Treg evaluation in FM3A cell transplantation model Effector Treg cells (CD4 ⁺ FoxP3 ⁺ KLRG1 ⁺ ) changes were evaluated. As a result, in both mNS-mFa55 and SW1208-mFa55, at doses of 1 mg/kg or more, the proportion of effector Tregs decreased to less than 0.2% of CD45-positive cells (Figure 12).

(3-7-8) Evaluation of systemic effects of spleen in FM3A cell transplant model
Changes in activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) in the spleen upon administration of mNS-mFa55 and SW1208-mFa55 were evaluated by FACS analysis. As a result, when mNS-mFa55 was administered, the ratio of activated helper T cells to CD45-positive cells in the spleen significantly increased, but SW1208-mFa55 decreased the number of activated helper T cells in the spleen even when the dose was increased. There was no significant increase in the ratio of CD45-positive cells (Figure 13). While the switch antibody exhibited efficacy equivalent to that of the control antibody, it was confirmed that it did not cause a reaction in tissues other than the tumor, and the concept that it was active only in the tumor local area was proven in vivo in mice.

[Example 4] Production of modified CTLA4 antibody and evaluation of its activity Further modification and evaluation of the anti-CTLA4 switch antibody produced in Example 3 was carried out.

(4-1) Enhancement of binding ability to CTLA4 by modification to enhance binding to ATP From the results of the structural analysis carried out in Example 2-1, it was found that CDR2 of the antibody heavy chain interacts with the phosphate group of AMP. It was shown that there is. When the small molecule is ATP, it was thought that the γ-phosphate group might cause steric hindrance with the heavy chain CDR2, so amino acid substitutions in this region were considered to enhance the ability to bind to ATP. Specifically, 04H1389-G1m/04L1086-lam1 in which R53Q and G55H modifications were introduced into the heavy chain variable region of 04H1207-G1m/04L1086-lam1 and 04H1208-G1m/04L1086-lam1 produced in Example 3. And 04H1382-G1m/04L1086-lam1 was created. In addition, 04H1389-G1m/04L1305-k0MT was created in which the light chain of 04H1389-G1m/04L1086-lam1 was replaced with the human κ chain sequence. Table 14 lists the sequence numbers of the heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, and Hyper Variable Region of these antibodies.

Amino acid sequences of heavy chains, light chains, and their hypervariable regions (indicated by SEQ ID NO.)

The binding of the produced variants to ATP and human CTLA4 was evaluated using Biacore T200. Binding measurements for ATP were carried out at 37°C using 20mM ACES (pH 7.4), 150mM NaCl, _2mM MgCl2, 0.05% Tween20 as a running buffer. First, antibodies were captured by interacting an antibody solution prepared with a running buffer with a chip in which Sure Protein A (GE Healthcare) was immobilized on a Series S Sensor Chip CM3 (GE Healthcare). Next, the binding ability with the antibody was evaluated by interacting with an ATP solution prepared in a running buffer. The chip was regenerated with 25 mM NaOH and 10 mM Glycine-HCl (pH 1.5), and antibodies were captured and measured repeatedly. The binding amount of each antibody to ATP is calculated by correcting the binding amount when ATP is injected at a concentration of 100 nM by the amount of antibody captured on the chip surface, and the binding amount of ATP per unit antibody amount is calculated. Ta. Binding measurements to human CTLA4 were performed using Biacore T200 as described in Example 3-3. Table 15 shows the results of these measurements.

Binding analysis to ATP and human CTLA4

04H1389-G1m/04L1086-lam1 and 04H1382-G1m/04L1086-lam1 have enhanced ATP binding ability compared to parent antibodies 04H1207-G1m/04L1086-lam1 and 04H1208-G1m/04L1086-lam1 before R53Q/G55H introduction. It had been. The binding ability of 04H1389-G1m/04L1086-lam1 and 04H1382-G1m/04L1086-lam1 to human CTLA4 was compared with that of the parent antibodies 04H1207-G1m/04L1086-lam1 and 04H1208-G1m/04L1086-lam1 before introduction of R53Q/G55H. It was enhanced approximately 10 times in the presence of 10 μM ATP. Comparison of binding amounts showed that the ability to bind to human CTLA4 was enhanced at lower ATP concentrations. 04H1389-G1m/04L1305-k0MT, in which the light chain of 04H1389-G1m/04L1086-lam1 was replaced with the human κ chain sequence, also has the same ATP binding ability as 04H1389-G1m/04L1086-lam1 and has ATP-dependent human CTLA4. was shown to have the binding ability of

(4-2) Preparation and evaluation of binding ability of anti-human CTLA4 control antibody and anti-CTLA4 switch antibody mFa55/04L1305-mk1 (abbreviation: SW1389-mFa55) was produced. The hNS-mFa55 antibody uses the heavy chain variable region MDX10D1H (SEQ ID NO: 26) and the light chain variable region MDX10D1L (SEQ ID NO: 27), and the constant region is the mouse heavy chain constant region mFa55 (SEQ ID NO: 18) and the wild type Mouse light chain constant region mk1 (SEQ ID NO: 19) is used. At this time, a mouse heavy chain constant region modified to enhance binding to Fcγ receptors was used and expressed and purified by a method known to those skilled in the art.
SW1389-mFa55 antibody uses heavy chain variable region 04H1389 (SEQ ID NO: 29) and light chain variable region 04L1305 (SEQ ID NO: 30), and the constant region is mouse heavy chain constant region mFa55 (SEQ ID NO: 18) and wild type Mouse light chain constant region mk1 (SEQ ID NO: 19) is used. At this time, a mouse heavy chain constant region modified to enhance binding to Fcγ receptors was used and expressed and purified by a method known to those skilled in the art.

The binding of the produced hNS-mFa55 and SW1389-mFa55 to human CTLA4 was evaluated. The running buffer used was 20mM ACES (pH 7.4), 150mM NaCl, 2mM _MgCl2 , 0.05% Tween20 with ATP added to the desired concentration, and measurements were performed at 37°C. . First, an antibody solution prepared in a running buffer that does not contain ATP is allowed to interact with a chip in which Rabbit Anti-Mouse IgG (Thermo Fisher Scientific) is immobilized on a Series S Sensor Chip CM5 (GE Healthcare). Captured. Next, by interacting a human CTLA4 solution prepared in a running buffer with ATP added or a human CTLA4 solution prepared in a running buffer without ATP to the desired concentration, in the presence of ATP and The binding ability of the antibody to human CTLA4 in the absence of ATP was evaluated. The chip was regenerated with 25 mM NaOH and 10 mM Glycine-HCl (pH 1.5), and antibodies were captured and measured repeatedly. The dissociation constant of each antibody for CTLA4 was calculated using Biacore T200 Evaluation Software 2.0. Specifically, the binding rate constant ka (L/mol/s) and dissociation rate constant kd (1/s) are calculated by global fitting the sensorgram obtained from the measurement using a 1:1 Langmuir binding model. The dissociation constant KD (mol/L) was calculated from that value. Table 16 shows the results of these measurements.

Binding analysis of variants with mouse constant region to human CTLA4

Both antibodies made with mouse constant regions were confirmed to bind to human CTLA4. SW1389-mFa55 was also shown to bind to human CTLA4 in an ATP-dependent manner, equivalent to 04H1389-G1m/04L1305-k0MT, which was constructed with the same variable region and human constant region listed in Table 15.

(4-3) Human CTLA4 knock-in, drug efficacy of anti-CTLA4 switch antibody and anti-CTLA4 non-switch antibody in a syngeneic tumor cell transplantation model using human CD3 transgenic mice, increase and decrease of Treg cells in the tumor , and movement of systemic reaction markers in the spleen (4-3-1) cell line
Hepa1-6/hGPC3 cells were used. This cell line was obtained by purchasing mouse liver cancer cell line Hepa1-6 from ATCC, constantly expressing the human Glypican 3 (hGPC3, SEQ ID NO: 181) gene by transfection, and cloning the cell line. Hepa1-6/hGPC3 cells were maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (SIGMA) and 600 μg/mL GENETICIN (gibco).

(4-3-2) Preparation of mouse model transplanted with syngeneic tumor line
human CTLA4 KI, which is a cross between a human CTLA4 knock-in mouse (Blood (2005) 106(9): 3127-3133) and an in-house produced human CD3 EDG replaced mouse (Sci Rep (2017) 7: 45839). Human CD3 EDG replaced mice (hCTLA4 KI hCD3 EDG replaced mice) were used. Hepa1-6/hGPC3 cells were subcutaneously transplanted into hCTLA4 KI hCD3 EDG replaced mice, and the model was established when the average volume of the transplanted tumor increased from approximately 200 mm ³ to 400 mm ³ .

The volume of the transplanted tumor was calculated using the following formula.
Tumor volume = long axis x short axis x short axis/2

(4-3-3) Preparation of drug to be administered
The drugs to be administered to the Hepa1-6/hGPC3 cell transplant model were the anti-CTLA4 switch antibody (SW1389-mFa55) prepared in Example 4-2 or the anti-human CTLA4 control antibody (hNS-mFa55) at 0.01 and 0.1, respectively. , 1, 5, 10, 20 mg/mL, 0.01, 0.1, 1, 3 mg/mL using His buffer (150 mM NaCl/20 mM His-HCl buffer pH 6.0).

(4-3-4) Drug administration when measuring antitumor effect SW1389-mFa55 at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg, and hNS-mFa55 at 0.1 mg/kg on the 7th day after transplantation. It was administered to mice via the tail vein at mg/kg, 1 mg/kg, 10 mg/kg, and 30 mg/kg. Table 17 shows details regarding drug treatment when measuring antitumor effects.

Antitumor effect measurement in Hepa1-6/hGPC3 cell transplant model

(4-3-5) Evaluation of antitumor effect The antitumor effect was evaluated using the tumor volume calculated using the formula described in (4-3-2). JMP 11.2.1 (SAS Institute Inc.) was used for statistical analysis.

The TGI (tumor growth inhibition) value was calculated using the following formula.
TGI = (1 - (average tumor volume of the group of interest at the time of measurement - average tumor volume at the time before antibody administration) ÷ (average tumor volume in the control group at the time of measurement - time before antibody administration) Average tumor volume at )) × 100

As a result, both hNS-mFa55 and SW1389-mFa55 showed efficacy of TGI=60 or more on the 18th day after administration at a dose of 1 mg/kg or more (Figures 14 and 15).

(4-3-6) Drug administration for evaluation of intratumoral Treg cells and verification of systemic effects in the spleen On the 7th day after transplantation, SW1389-mFa55 was administered at 0.1 mg/kg, 1 mg/kg, 10 mg/kg. , 100 mg/kg, 500 mg/kg were administered to mice through the tail vein, and hNS-mFa55 was administered through the tail vein at 0.1 mg/kg, 1 mg/kg, 10 mg/kg, 30 mg/kg. . Details regarding drug treatment during evaluation of intratumoral Treg cells and verification of systemic effects in the spleen are shown in Table 18.

Verification of intratumoral and systemic effects in Hepa1-6/hGPC3 cell transplantation model (hNS-mFa55 and SW1389-mFa55)

(4-3-7) Removal of tumor and spleen from model mouse transplanted with Hepa1-6/hGPC3 cells Six days after antibody administration, the mouse was euthanized under anesthesia, and the tumor and spleen were removed. Prepare a cell suspension from the removed spleen using RPMI-1640 medium (SIGMA) containing 10% FBS (SIGMA), then perform hemolysis using the Mouse Erythrocyte Lysing kit (R&D) to prepare spleen cells. did. The removed tumor was crushed using a Tumor dissociation kit, mouse (miltenyi). Both spleen cells and crushed tumors were reacted with the following antibodies, and the fraction of immune cells present was analyzed by FACS analysis. Anti-CD45 antibody (BD, clone: 30-F11), anti-CD3 antibody (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RM4-5), anti-FoxP3 antibody (eBioscience, clone: FJK-16s), Anti-ICOS antibody (eBioscience, clone: 7E17G9), anti-CCR7 antibody (Biolegend, clone: 4B12), anti-KLRG1 antibody (Biolegend, clone: 2F1/KLRG1). FACS analysis was performed on a BD LSRFortessa X-20 (BD).

(4-3-8) Tumor Treg evaluation in Hepa1-6/hGPC3 cell transplant model
Changes in intratumoral effector Treg cells (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1 ⁺ ) were evaluated upon administration of SW1389-mFa55 and hNS-mFa55. As a result, in both hNS-mFa55 and SW1389-mFa55, at doses of 1 mg/kg or more, the proportion of effector Tregs decreased to less than 0.2% of CD45-positive cells (Figure 16).

(4-3-9) Evaluation of systemic effects of spleen in Hepa1-6/hGPC3 cell transplant model
Changes in activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) in the spleen upon administration of hNS-mFa55 or SW1389-mFa55 were evaluated by FACS analysis. As a result, when hNS-mFa55 was administered, the ratio of activated helper T cells to CD45-positive cells in the spleen significantly increased, but when SW1389-mFa55 was administered, the ratio of activated helper T cells in the spleen remained significant even when the dose was increased. No significant increase was observed (Figure 17). While the switch antibody exhibited drug efficacy equivalent to that of the control antibody, it was confirmed that it did not cause a reaction in tissues other than the tumor, and the concept that it was active only in the tumor local area was proven in vivo in human CTLA4 KI mice.

(4-4) Preparation of anti-CTLA4 control antibody and anti-CTLA4 switch antibody for cynomolgus monkey toxicity test Anti-CTLA4 control antibody (MDX10D1H-Kn125/MDX10D1L-k0MT//MDX10D1H-Hl076/MDX10D1L-k0MT Abbreviation: NS-ART1) And an anti-CTLA4 switch antibody (04H1389-Kn125/04L1305-k0MT//04H1389-Hl076/04L1305-k0MT abbreviation: SW1389-ART1) was produced. The NS-ART1 antibody uses the heavy chain variable region MDX10D1H (SEQ ID NO: 26) and the light chain variable region MDX10D1L (SEQ ID NO: 27), and the constant region is the human heavy chain constant region Kn125 (SEQ ID NO: 26) described in prior patent WO2013/002362. SEQ ID NO: 31), human heavy chain constant region Hl076 (SEQ ID NO: 32), and human light chain constant region k0MT (SEQ ID NO: 33) are used. At this time, a human heavy chain constant region modified to enhance binding to Fcγ receptors was used and expressed and purified by a method known to those skilled in the art.
SW1389-ART1 antibody uses heavy chain variable region 04H1389 (SEQ ID NO: 29) and light chain variable region 04L1305 (SEQ ID NO: 30), and the constant region is human heavy chain constant region Kn125 (SEQ ID NO: 31) and human heavy chain variable region 04L1305 (SEQ ID NO: 30). Chain constant region Hl076 (SEQ ID NO: 32) and human light chain constant region k0MT (SEQ ID NO: 33) are used. At this time, a human heavy chain constant region modified to enhance binding to Fcγ receptors was used and expressed and purified by a method known to those skilled in the art.
Note that the heterodimeric antibodies (antibodies having two different heavy chain polypeptides and/or two different light chain polypeptides) in this specification are named according to the following rules; heavy chain variable region) - (first heavy chain constant region) / (first light chain variable region) - (first light chain constant region) // (second heavy chain variable region) - (first (second heavy chain constant region)/(second light chain variable region)-(second light chain constant region). For example, if the antibody name is 04H1389-Kn125/04L1305-k0MT//04H1389-Hl076/04L1305-k0MT, the first heavy chain variable region of this antibody is 04H1389, the first heavy chain constant region is Kn125, and the first heavy chain constant region is Kn125. The light chain variable region of is 04L1305, the first light chain constant region is k0MT, the second heavy chain variable region is 04H1389, the second heavy chain constant region is Hl076, the second light chain variable region is 04L1305, the second means that the light chain constant region of is k0MT.

(4-5) Implementation of cynomolgus monkey toxicity test For the purpose of evaluating and comparing toxicity including systemic reactions, the NS-ART1 antibody and SW1389-ART1 antibody prepared in Example 4-4 were tested in male cynomolgus monkeys (3 each Example: 60 mg/kg was administered once every week for a total of 5 times. Administration was performed by low-speed intravenous administration using a syringe pump, and general conditions were observed, weight measurements, blood and blood chemistry tests, bone marrow tests, pathological tests, and plasma drug concentration measurements were performed.
Anti-drug antibodies were observed for both antibodies during the administration period, but exposure was maintained until the end of the administration period. Systemic autoimmune disease-like changes were observed upon administration of NS-ART1 antibody, including the expression of autoantibodies, inflammatory changes (vasculitis, inflammatory cell infiltration), anemic changes, and activation of T cells; -The above-mentioned changes were not observed with ART1 antibody administration. Therefore, these results showed that the SW1389-ART1 antibody reduces toxicity in living organisms.

[Example 5] Production of modified CTLA4 antibody and evaluation of its activity Further modification and evaluation of the anti-CTLA4 switch antibody produced in Example 4 was carried out.

(5-1) Antibody optimization by comprehensive modification introduction and Framework replacement
Amino acid modifications were comprehensively introduced into the CDRs of 04H1389-G1m/04L1086-lam1, and modifications with better profiles were searched for. Comprehensive introduction and evaluation of amino acid modifications were performed using the method described in Example 3-3. A combination of the modifications found here and a modification that replaced the Framework was created. Table 19 lists the sequence numbers of the heavy chain variable region, light chain variable region, heavy chain constant region, light chain constant region, and Hyper Variable Region of these antibodies. Light chains 04L1594-lam1, 04L1581-lam1, 04L1610-lam1, 04L1612-lam1, and 04L1610-lam1 in Table 19 have CDR and framework modifications introduced to the light chain 04L1086-lam1 of the parent antibody, and human It has a germline sequence framework and constant region of the λ chain. Furthermore, 04L1615-k0MT, 04L1616-k0MT, and 04L1617-k0MT have CDR modifications introduced to 04L1086-lam1, and have the framework and constant region of the human κ chain germline sequence. Heavy chain variable region 04H1389v373 has modifications introduced to the CDRs of heavy chain variable region 04H1389 of the parent antibody, and 04H1637, 04H1643, 04H1654, 04H1656, 04H1642, 04H1735 have modifications introduced to the CDRs of 04H1389, and This is a heavy chain variable region with the sequence replaced with a different germline.

Amino acid sequences of heavy chains, light chains, and their hypervariable regions (indicated by SEQ ID NO.)

The binding activity of the produced variants to human CTLA4 was evaluated by the method described in Example 3-3 (Table 20).

Binding analysis to human CTLA4

KD values marked with * in the table were calculated using a steady state model. All variants produced using 04H1389-G1m/04L1086-lam1 as a parent antibody bind to human CTLA4 in an ATP-dependent manner and exhibit a KD of 3.7×10 ^-8 M in the presence of 10 μM ATP. It was shown to have stronger binding ability than antibodies. Furthermore, it was shown that all of these antibodies had stronger binding ability than the existing anti-human CTLA4 antibody MDX10D1H-G1m/MDX10D1L-k0MT in the presence of 10 μM ATP.

The binding ability of the produced variant to human CTLA4 in the presence of ADP and AMP was evaluated using Biacore T200, and compared with the binding ability in the presence of ATP. The binding ability to human CTLA4 in the presence of ADP and AMP was determined using the method described in Example 3-3, similar to the evaluation of the binding ability in the presence of ATP. Alternatively, it was performed using AMP (Table 21).

Evaluation of ATP, ADP and AMP dependence

While existing human CTLA4 antibodies MDX10D1H-G1m/MDX10D1L-k0MT showed similar kinetic parameters regardless of the type of small molecule or the presence or absence of small molecules, ATP-dependent anti-CTLA4 antibodies It also binds to human CTLA4 not only in the presence of ATP but also in the presence of ADP and AMP, and the binding ability in the presence of these small molecules is greater than that in the absence of small molecules shown in Table 20. it was high. Therefore, these antibodies were shown to bind to CTLA4 in an ATP-, ADP-, and AMP-dependent manner. These antibodies had the highest binding ability in the presence of ATP, followed by the highest binding ability in the presence of ADP, and the lowest binding ability in the presence of AMP. In both cases, the binding ability in the presence of ADP was about 3 times stronger than in the presence of AMP, and the binding ability in the presence of ATP was about 5 times stronger than in the presence of AMP. . The ka values were similar in the presence of both small molecules, but there were differences in the kd values, with more dissociation in the presence of ADP than in the presence of ATP, and more in the presence of AMP than in the presence of ADP. The dissociation was rapid, indicating that the difference in KD values depending on the type of small molecule is due to the difference in dissociation rate.

Next, several variants were evaluated for their ability to bind to mouse CTLA4 and cynomolgus monkey CTLA4. The binding activity to human CTLA4, mouse CTLA4, and cynomolgus monkey CTLA4 was evaluated using Biacore T200 by the method described in Example 3-3 (Table 22). Cynomolgus monkey CTLA4 was prepared by the following method.
CyCTLA4-His-BAP (SEQ ID NO: 50), in which a His tag and a BAP tag were fused to the C-terminus of the extracellular region of cynomolgus monkey CTLA4, was gene synthesized and inserted into an animal expression plasmid. Prepared for human embryonic kidney cell-derived FreeStyle 293-F strain (Invitrogen) suspended in FreeStyle 293 Expression Medium medium (Invitrogen) at a cell density of 1.33 × 10 ⁶ cells/mL and seeded in flasks. The plasmid was introduced by lipofection, and 3 hours after the plasmid introduction, biotin was added to a final concentration of 100 μM, and cultured in a CO ₂ incubator (37° C., 8% CO ₂ , 125 rpm) for 4 days. The antigen was purified from the culture supernatant of each specimen by a method known to those skilled in the art. The absorbance of the purified antigen solution at 280 nm was measured using a spectrophotometer. The concentration of the purified antigen was calculated from the obtained measured value using the extinction coefficient calculated by the PACE method (Protein Science (1995) 4, 2411-2423).

Binding analysis for human, mouse, and cynomolgus CTLA4

All six small molecule-dependent CTLA4 antibodies evaluated were shown to bind not only to human CTLA4 but also to mouse CTLA4 and cynomolgus monkey CTLA4 in an ATP-dependent manner.

(5-2) Production of modified anti-CTLA4 switch antibody and negative control antibody Modified anti-CTLA4 switch antibody (04H1654-mFa55m2P1/04L1610-ml0r//04H1656-mFa55m2N1/04L1610-ml0r Abbreviation: SW1610-mFa55, 04H1654 -mFa55m2P1/04L1612-ml0r//04H1656-mFa55m2N1/04L1612-ml0r Abbreviation: SW1612-mFa55, and 04H1389-mFa55/04L1615-mk1 Abbreviation: SW1615-mFa55), and negative control antibody (IC17Hdk-mFa5 5/IC17L-mk1 Abbreviation: KLH-mFa55) was created.
SW1615-mFa55 antibody uses heavy chain variable region 04H1389 (SEQ ID NO: 29) and light chain variable region 04L1615 (SEQ ID NO: 34), and the constant region is mouse heavy chain constant region mFa55 (SEQ ID NO: 18) and wild type Mouse light chain constant region mk1 (SEQ ID NO: 19) is used. At this time, a mouse heavy chain constant region modified to enhance binding to Fcγ receptors was used and expressed and purified by a method known to those skilled in the art.
The SW1610-mFa55 antibody has one heavy chain variable region 04H1654 (SEQ ID NO: 35) as a constant region, mouse heavy chain constant region mFa55m2P1 (SEQ ID NO: 36), and the other heavy chain variable region 04H1656 (SEQ ID NO: 37) as a constant region. ) is linked with the mouse heavy chain constant region mFa55m2N1 (SEQ ID NO: 38), and the light chain variable region 04L1610 (SEQ ID NO: 39) is linked with the wild type mouse light chain constant region ml0r (SEQ ID NO: 22). It was expressed and purified using methods known to those skilled in the art.
The SW1612-mFa55 antibody has one heavy chain variable region 04H1654 (SEQ ID NO: 35) as a constant region, mouse heavy chain constant region mFa55m2P1 (SEQ ID NO: 36), and the other heavy chain variable region 04H1656 (SEQ ID NO: 37) as a constant region. ) is linked with the mouse heavy chain constant region mFa55m2N1 (SEQ ID NO: 38), and the light chain variable region 04L1612 (SEQ ID NO: 40) is linked with the wild type mouse light chain constant region ml0r (SEQ ID NO: 22), It was expressed and purified using methods known to those skilled in the art.
In the negative control antibody, the heavy chain variable region IC17Hdk (SEQ ID NO: 51) is linked to the mouse heavy chain constant region mFa55 (SEQ ID NO: 18) as a constant region, and the light chain variable region IC17L (SEQ ID NO: 52) is linked to the wild It was expressed and purified using a type mouse light chain constant region mk1 (SEQ ID NO: 19) by a method known to those skilled in the art.

(5-3) Evaluation of the binding ability of an antibody having a mouse constant region to human CTLA4 The binding ability of an anti-CTLA4 antibody having a mouse constant region to an antigen was evaluated by the method described in Example 4-2 (Table twenty three). All of these antibodies with mouse constant regions were shown to have the same ability to bind to human CTLA4 in an ATP-dependent manner as the antibodies listed in Table 22 with the same variable region and human constant regions.

Binding analysis of variants with mouse constant region to human CTLA4

(5-4) Human CTLA4 knock-in, efficacy of anti-CTLA4 switch antibody in a syngeneic tumor cell transplantation model using human CD3 transgenic mice, increase and decrease of Treg cells in the tumor, and movement of systemic reaction markers in the spleen (5-4) 4-1) Cell lines
Hepa1-6/hGPC3 cells were used. This cell line was obtained by purchasing the mouse liver cancer cell line Hepa1-6 from ATCC, constantly expressing the human Glypican 3 (hGPC3) gene by transfection, and cloning it. Hepa1-6/hGPC3 cells were maintained and passaged in D-MEM (high glucose) medium (SIGMA) containing 10% FBS (SIGMA) and 0.6 mg/mL G418 (Nacalai Tesque).

(5-4-2) Preparation of mouse model transplanted with syngeneic tumor line
human CTLA4 KI, which is a cross between a human CTLA4 knock-in mouse (Blood (2005) 106(9): 3127-3133) and an in-house produced human CD3 EDG replaced mouse (Sci Rep (2017) 7: 45839). Human CD3 EDG replaced mice (hCTLA4 KI hCD3 EDG replaced mice) were used. Hepa1-6/hGPC3 cells were subcutaneously transplanted into hCTLA4 KI hCD3 EDG replaced mice, and the model was established when the average volume of the transplanted tumor increased from approximately 200 mm ³ to 400 mm ³ .

The volume of the transplanted tumor was calculated using the following formula.
Tumor volume = long axis x short axis x short axis/2

(5-4-3) Preparation of drug to be administered
The drugs administered to the Hepa1-6/hGPC3 cell transplant model were the anti-CTLA4 switch antibodies (SW1610-mFa55, SW1612-mFa55, SW1615-mFa55) prepared in Example 5-2. The drugs to be administered were prepared at concentrations of 0.03 mg/mL, 0.1 mg/mL, and 0.3 mg/mL using His-buffer (20 mM His-HCl, 150 mM NaCl, pH 6.0).

(5-4-4) Drug administration for anti-tumor effect measurement Eight days after transplantation, three anti-CTLA4 switch antibody samples were administered to mice via the tail vein at 0.3 mg/kg, 1 mg/kg, and 3 mg/kg, respectively. administered. Table 24 shows details regarding drug treatment when measuring antitumor effects.

Measurement of antitumor effect in Hepa1-6/hGPC3 cell transplant model (anti-CTLA4 switch antibody)

(5-4-5) Evaluation of antitumor effect The antitumor effect was evaluated using the tumor volume calculated using the formula described in (5-4-2).

The tumor growth inhibition rate (TGI) value was calculated using the following formula.
TGI (%) = (1 - (average tumor volume of the group of interest at the time of measurement - average value of tumor volume of the group of interest at the time of first administration) ÷ (average tumor volume of the control group at the time of measurement - Average tumor volume in the control group at the time of first administration)) × 100

As a result, SW1610-mFa55 and SW1612-mFa55 showed efficacy of TGI=60% or more on the 16th day after administration at a dose of 1 mg/kg or more, and SW1615-mFa55 at a dose of 3 mg/kg or more (Figure 18 to Figure 20).

(5-4-6) Drug administration for evaluation of intratumoral Treg cells and verification of systemic effects in the spleen On day 10 after transplantation, SW1610-mFa55 was administered at 50 mg/kg, 100 mg/kg, 200 mg/kg. , SW1612-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, SW1615-mFa55 at 50 mg/kg, 100 mg/kg, 200 mg/kg, 400 mg/kg were administered to mice via the tail vein. did. Furthermore, a negative control antibody, IC17Hdk-mFa55/IC17L-mk1 (abbreviation: KLH-mFa55), was administered to the control group at 400 mg/kg via the tail vein. Details regarding drug treatment for evaluation of intratumoral Treg cells and verification of systemic effects in the spleen are shown in Table 25.

Verification of intratumoral and systemic effects in Hepa1-6/hGPC3 cell transplantation model (anti-CTLA4 switch antibody)

(5-4-7) Removal of tumor and spleen from model mouse transplanted with Hepa1-6/hGPC3 cells Six days after antibody administration, the mouse was euthanized under anesthesia, and the tumor and spleen were removed. Prepare a cell suspension from the removed spleen using RPMI-1640 medium (SIGMA) containing 10% FBS (SIGMA), then perform hemolysis using the Mouse Erythrocyte Lysing kit (R&D) to prepare spleen cells. did. The removed tumor was crushed using a Tumor dissociation kit, mouse (miltenyi). Both spleen cells and crushed tumors were reacted with the following antibodies, and the fraction of immune cells present was analyzed by FACS analysis. Anti-CD45 antibody (BD, clone: 30-F11), anti-CD3 antibody (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RM4-5), anti-FoxP3 antibody (eBioscience, clone: FJK-16s), Anti-ICOS antibody (eBioscience, clone: 7E17G9), anti-CCR7 antibody (Biolegend, clone: 4B12), anti-KLRG1 antibody (Biolegend, clone: 2F1/KLRG1). FACS analysis was performed on a BD LSRFortessa X-20 (BD).

(5-4-8) Tumor Treg evaluation in Hepa1-6/hGPC3 cell transplant model Changes in effector Treg cells (CD4 ⁺ FoxP3 ⁺ CCR7 ^low KLRG1 ⁺ ) within the tumor upon administration of anti-CTLA4 switch antibody were evaluated. As a result, the proportion of effector Tregs decreased to less than 0.2% of CD45-positive cells in all doses of SW1610-mFa55, SW1612-mFa55, and SW1615-mFa55 administered (FIG. 21).

(5-4-9) Evaluation of systemic effects on the spleen in a Hepa1-6/hGPC3 cell transplant model Changes in activated helper T cells (CD4 ⁺ Foxp3 ^- ICOS ⁺ ) in the spleen upon administration of anti-CTLA4 switch antibody were evaluated using FACS. Evaluated by analysis. As a result, SW1610-mFa55 and SW1612-mFa55 activated helper T cells in the spleen at a dose of 50 mg/kg, and SW1615-mFa55 at a dose of 200 mg/kg or less. There was no significant increase in the ratio of CD45-positive cells to CD45-positive cells. For the significance test, JMP 11.2.1 (SAS Institute Inc.) was used, and Dunnett's test was performed on the KLH-mFa55 administration group (FIG. 22). While all of the switch antibodies exhibited medicinal efficacy, it was confirmed that they did not cause a reaction in tissues other than the tumor, and it was confirmed that they had the property of exhibiting activity only in the tumor local area.

[Example 6] Production of modified Fc that enables enhancement of ADCC/ADCP activity In order to produce antibodies with enhanced ADCC and ADCP, which are cytotoxic effector functions, the ability to bind to FcγRIIIa and FcγRIIa, which are active FcγRs, is enhanced. The creation of modified Fc regions was investigated.

(6-1) Preparation and evaluation of variants with enhanced binding to FcγR
Heterodimeric antibody, 04H1637-, which has heavy chain constant regions Kn125 and Hl076 with enhanced binding ability to FcγR, 04H1637 as the heavy chain variable region, and 04L1610-lam1 as the light chain, described in WO2013/002362 Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1 was created. Specifically, one heavy chain variable region has 04H1637 (SEQ ID NO: 138), and G1d (SEQ ID NO: 158) in which the C-terminal Gly and Lys of the human IgG1 heavy chain constant region have been removed , L234Y/L235Q/G236W/S239M/H268D/D270E/S298A were introduced, and the gene for antibody heavy chain 04H1637-Kn125 (SEQ ID NO: 162) was created, which has a modified Y349C/T366W that promotes heterodimerization in the CH3 region. It was done. Similarly, the other heavy chain variable region has 04H1637 (SEQ ID NO: 138), and D270E/K326D/A330M/K334E are introduced into human IgG1 heavy chain constant region G1d (SEQ ID NO: 158), Furthermore, a gene for antibody heavy chain 04H1637-Hl076 (SEQ ID NO: 163) was created, which has modifications D356C/T366S/L368A/Y407V in the CH3 region that promote heterodimerization. Using 04L1610-lam1 (SEQ ID NO: 161) as the antibody light chain, the heterodimer 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1 was produced by a method known to those skilled in the art. Among the modifications introduced in the CH2 region here, in addition to L235Q, G236W, S239M, H268D, D270E, S298A, K326D, and K334E, L234F, which was reported in WO2013/002362 as a modification that changes binding to FcγR , A330K, Mol. Cancer Ther., 2008, 7, 2517-2527 and G236A, I332E, I332D reported in WO2004/029207, T250V, T307P reported as modifications to improve stability in WO2013/118858. Combined antibody heavy chains 04H1637-Kn462 (SEQ ID NO: 164), 04H1637-Hl441 (SEQ ID NO: 165), 04H1637-Hl445 (SEQ ID NO: 166), 04H1637-Kn461 (SEQ ID NO: 167), 04H1637-Hl443 (SEQ ID NO: 167) The gene number: 168) was created. In addition, Gly and Lys at the C-terminus of human IgG1 (IGHG1*03) are removed, the CH2 region has the same modification as Kn462, and the CH3 region has the modification E356K that promotes heterodimerization as described in WO2006/106905. Then, a gene for antibody heavy chain 04H1654-KT462 (SEQ ID NO: 182) having 04H1654 (SEQ ID NO: 140) as the heavy chain variable region was created. Similarly, Gly and Lys at the C-terminus of human IgG1 (IGHG1*03) were removed, the CH2 region had the same modification as Hl441, and the CH3 region had the modification K439E that promotes heterodimerization described in WO2006/106905. The gene for the antibody heavy chain 04H1656-HT441 (SEQ ID NO: 170) having 04H1656 (SEQ ID NO: 141) as the heavy chain variable region was created. Similarly, genes 04H1656-HT445 (SEQ ID NO: 171), 04H1654-KT461 (SEQ ID NO: 183), and 04H1656-HT443 (SEQ ID NO: 173) were produced. Furthermore, combinations of modifications that improve blood dynamics of antibodies described in Mab, 2017, 9, 844-853 were investigated. Specifically, the CH3 region of 04H1654-KT462 (SEQ ID NO: 182) was modified to N434A/Y436T/ which is a combination of modifications that enhance binding to human FcRn under acidic conditions and modifications that reduce binding to Rheumatoid factor. The gene 04H1654-KT473 (SEQ ID NO: 184) into which Q438R/S440E was introduced was created. Similarly, a gene of 04H1656-HT482 (SEQ ID NO: 185) was created in which N434A/Y436T/Q438R/S440E were introduced into 04H1656-HT445 (SEQ ID NO: 171). Similarly, antibody heavy chain 04H1654-KT481 (SEQ ID NO: 186) and antibody heavy chain 04H1656-HT498 (SEQ ID NO: 187) were created by introducing the same modifications to 04H1654-KT461 and 04H1656-HT443, respectively. By combining these heavy chains and using 04L1610-lam1 or 04L1612-lam1 (SEQ ID NO: 188) as the light chain, the desired heterodimeric antibody was produced.

The extracellular domain of FcγR was prepared by the following method. First, the gene for the extracellular domain of FcγR was synthesized by a method known to those skilled in the art. At that time, the sequence of each FcγR was created based on information registered with NCBI. Specifically, for FcγRI, the sequence is NCBI accession # NM_000566.3, for FcγRIIa, the sequence is NCBI accession # NM_001136219.1, for FcγRIIb, the sequence is NCBI accession # NM_004001.3, and for FcγRIIIa, the sequence is NCBI accession # NM_004001.3. # Created based on the sequence of NM_001127593.1, with a His tag added to the C-terminus. In addition, the polymorphic site of FcγRIIa was created with reference to J. Exp. Med., 1990, 172, 19-25, and the polymorphic site of FcγRIIIa was created with reference to J. Clin. Invest., 1997, 100, 1059-1070. It was done. An expression vector was created by inserting the obtained gene fragment into an animal cell expression vector. The prepared expression vector was transiently introduced into human embryonic kidney cancer cell-derived FreeStyle 293 cells (Invitrogen), and the target protein was expressed. After the culture supernatant was collected, it was passed through a 0.22 μm filter and purified using the following four steps. The first step is cation exchange column chromatography (SP Sepharose FF), the second step is affinity column chromatography for His tag (HisTrap HP), the third step is gel filtration column chromatography (Superdex200), and the fourth step is sterile Filtration was performed. However, for FcγRI, anion exchange column chromatography using Q sepharose FF was performed in the first step. The concentration of the purified protein was calculated by measuring the absorbance at 280 nm using a spectrophotometer and using the extinction coefficient calculated from the obtained value by a method such as PACE (Protein Science, 1995 , 4, 2411-2423). Human FcRn was prepared by the method described in WO2010/107110.

Interaction analysis between the produced antibody and human FcγR was performed using Biacore T200 in the following manner. The running buffer used was 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween20 (pH 7.4), and measurements were performed at 25°C. For the sensor chip, a chip with CaptureSelect Human Fab-lambda Kinetics Biotin Conjugate (Thermo Fisher Scientific) immobilized on Series S SA (GE Healthcare) was used. The antibody of interest was captured using this chip, and each FcγR diluted in running buffer was allowed to interact with it. The chip was regenerated with 10mM Glycine-HCl (pH 1.5) and 25mM NaOH, and repeated antibody captures and measurements were performed. The dissociation constant KD (mol/L) for FcγR of each antibody was calculated using Biacore T200 Evaluation Software 2.0, the dissociation constant for FcγRIa and FcγRIIIa was calculated using the 1:1 Langmuir binding model, and the dissociation constant for FcγRIIa was calculated using the Steady state affinity model. It was done. Furthermore, for FcγRIIb, the amount of FcγRIIb bound per unit amount of antibody was calculated by correcting the amount of FcγRIIb bound determined from the sensorgram obtained by measurement with the amount of antibody captured on the chip surface.

Interaction analysis between the produced antibody and human FcRn was performed using Biacore T200 in the following manner. The running buffer used was 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween20 (pH 6.0), and measurements were performed at 25°C. For the sensor chip, a chip with CaptureSelect Human Fab-lambda Kinetics Biotin Conjugate (Thermo Fisher Scientific) immobilized on Series S SA (GE Healthcare) was used. The antibody of interest was captured onto this chip and allowed to interact with FcRn diluted in running buffer. The chip was regenerated with 10 mM Glycine-HCl (pH 1.5) and 25 mM NaOH, and antibodies were captured and measured repeatedly. The dissociation constant of each antibody for FcRn was calculated using a Steady state model using Biacore T200 Evaluation Software 2.0.
Table 26 shows the results of these measurements.

Binding analysis of Fc region variants to human FcγR and FcRn

The values of "KD for hFcRn (M)" and "KD for hFcγRs (M)" in the table indicate the dissociation constants for hFcRn and each FcγR, respectively, and the "binding amount" is when FcγRIIb is interacted with at 1000 nM. The amount of FcγRIIb bound per unit amount of antibody is shown. "Relative value of G1m and hFcRn to KD" and "Relative value of G1m and hFcγRs to KD" are the values obtained by dividing the KD value of 04H1637-G1m/04L1610-lam1 for hFcRn and each FcγR by the KD value of each variant. "Relative binding amount" indicates the value obtained by dividing the binding amount of each variant to FcγRIIb by the binding amount of 04H1637-G1m/04L1610-lam1. The amino acid sequences of antibody heavy chain 04H1637-G1m and antibody light chain 04L1610-lam1 are shown in SEQ ID NOs: 160 and 161, respectively. It was shown that all of the prepared heterodimeric antibodies had enhanced binding to FcγRIIa and FcγRIIIa compared to 04H1637-G1m/04L1610-lam1, which has a constant region of natural human IgG1. Also 04H1637-Kn462/04L1610-lam1//04H1637-Hl441/04L1610-lam1, 04H1637-Kn462/04L1610-lam1//04H1637-Hl445/04L1610-lam1, 04H1637-Kn461/04L16 10-lam1//04H1637-Hl443/04L1610- lam1 has enhanced binding to FcγRIIa compared to 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1, which has the Fc region variant reported in WO2013/002362. Shown. In addition, the binding ability of 04H1637-Kn462/04L1610-lam1//04H1637-Hl445/04L1610-lam1 to FcγRIIIa is equivalent to that of 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1, and 637-Kn461/04L1610 It was shown that the binding ability of -lam1//04H1637-Hl443/04L1610-lam1 to FcγRIIIa was enhanced compared to that of 04H1637-Kn125/04L1610-lam1//04H1637-Hl076/04L1610-lam1. Similarly, 04H1654-KT462/04L1610-lam1//04H1656-HT445/04L1610-lam1 and 04H1654-KT462/04L1612-lam1//04H1656-HT445/ have different heterodimerization modifications in the constant and CH3 regions of IGHG1*03. 04L1610-lam1 is 04H1637-Kn462/04L1610-lam1//04H1637-Hl445/04L1610-lam1, 04H1654-KT461/04L1610-lam1//04H1656-HT443/04L1610-lam1 and 04H1654- KT461/04L1612-lam1//04H1656- HT443/04L1610-lam1 was shown to have a binding profile to FcγR comparable to 04H1637-Kn461/04L1610-lam1//04H1637-Hl443/04L1610-lam1. 04H1654-KT473/04L1610-lam1//04H1656-HT482/04L1610-lam1, 04H1654-KT481/04L1610-lam1//04H1656-HT498/04L1610-lam1, 04H1654-KT has also been modified to improve blood dynamics. 473/ 04L1612-lam1//04H1656-HT482/04L1612-lam1, 04H1654-KT481/04L1612-lam1//04H1656-HT498/04L1612-lam1 is more human-like than the antibody before the modification to improve blood dynamics was introduced. It was shown that the binding ability to FcRn was improved and the binding ability to FcγR was equivalent.

Furthermore, 04H1656-HT451 (sequence The gene number: 272) was created. The amino acid sequence of antibody heavy chain HT451 is shown in SEQ ID NO: 276. A heterodimeric antibody was created by combining 04H1654-KT473 and 04H1656-HT451 and using 04L1610-lam1 as the antibody light chain. Table 27 shows the results of interaction analysis between the produced antibodies and human FcRn and human FcγR.

Binding analysis of Fc region variants to human FcγR and FcRn

Both of the prepared heterodimeric antibodies 04H1654-KT462/04L1610-lam1//04H1656-HT441/04L1610-lam1 and 04H1654-KT473/04L1610-lam1//04H1656-HT451/04L1610-lam1 are equivalent to natural human IgG1. It was shown that binding to FcγRIIa and FcγRIIIa, which are active FcγRs, was enhanced compared to 04H1656-G1m/04L1610-lam1, which has a constant region. Furthermore, all of these antibodies maintained binding to FcγRIIb, which is an inhibitory FcγR, at the same level as 04H1656-G1m/04L1610-lam1. N434A/Y436T/Q438R/S440E was introduced for 04H1654-KT462/04L1610-lam1//04H1656-HT441/04L1610-lam1 04H1654-KT473/04L1610-lam1//04H1656-HT451/04L16 10-lam1 is before introduction In comparison, it was shown that the binding ability to human FcRn was enhanced.

Next, the binding activity to human FcRn and FcγR was evaluated for FcγR binding-enhancing variants produced using different heterodimerization modifications described in Nat. Biotechnol., 1998, 16, 677-681. It has 04H1389 (SEQ ID NO: 136) as the heavy chain variable region, and the same modification as KT462 is made in the CH2 region of the constant region in which the C-terminal Gly and Lys of human IgG1 (IGHG1*03) are removed as the heavy chain constant region. Antibody heavy chain 04H1389-Ks462 (SEQ ID NO: 191) in which T366W was introduced as a heterodimerization modification in the CH3 region, 04H1389-Km462 (SEQ ID NO: 199) in which Y349C/T366W was used as a heterodimerization modification A gene was created. Antibody heavy chain 04H1389-Hs445 (SEQ ID NO: 192) has the same modification as HT445 introduced into the CH2 region and T366S/L368A/Y407V as a heterodimerization modification into the CH3 region, and E356C/T366S as a heterodimerization modification. The gene 04H1389-Hm445 (SEQ ID NO: 200) was created using /L368A/Y407V. Similarly, 04H1389-Ks461 (SEQ ID NO: 193), 04H1389-Km461 (SEQ ID NO: 201), 04H1389-Hs443 (SEQ ID NO: 194), 04H1389-Hm443 (SEQ ID NO: 194), which has the same modification as KT461 and HT443 as CH2 region modification. No.: 202) was created. In addition, modifications N434A/Y436T/Q438R/S440E to improve blood dynamics were introduced into these antibody heavy chain constant regions Ks462, Hs445, Ks461, Hs443, Km462, Hm445, Km461, and Hm443, and the antibody has 04H389 as a variable region. Antibody heavy chain 04H1389-Ks473 (SEQ ID NO: 195), 04H1389-Hs482 (SEQ ID NO: 196), 04H1389-Ks481 (SEQ ID NO: 197), 04H1389-Hs498 (SEQ ID NO: 198), 04H1389-Km473 (SEQ ID NO: 203), 04H1389-Hm482 (SEQ ID NO: 204), 04H1389-Km481 (SEQ ID NO: 205), and 04H1389-Hm498 (SEQ ID NO: 206) were created. 04L1615-k0MT (SEQ ID NO: 190) was used as the light chain to create the desired heterodimer. In addition, a homodimer 04H1389-G1m/04L16150k0MT having 04H1389-G1m (SEQ ID NO: 189) was produced as a comparison target. Interaction analysis between the produced antibody and human FcγR was performed using Biacore T200. The running buffer used was 50 mM Na-Phosphate, 150 mM NaCl, and 0.05% Tween20 (pH 7.4), and measurements were performed at 25°C. The sensor chip used was a Series S SA (GE Healthcare) chip on which CaptureSelect Human Fab-kappa Kinetics Biotin Conjugate (Thermo Fisher Scientific) was immobilized. The antibody of interest was captured using this chip, and each FcγR diluted in running buffer was allowed to interact with it. The chip was regenerated with 10mM Glycine-HCl (pH 1.5) and 25mM NaOH, and repeated antibody captures and measurements were performed. The dissociation constant KD (mol/L) for FcγR of each antibody was calculated using Biacore T200 Evaluation Software 2.0, the dissociation constant for FcγRIa and FcγRIIIa was calculated using the 1:1 Langmuir binding model, and the dissociation constant for FcγRIIa was calculated using the Steady state affinity model. It was done. Furthermore, for FcγRIIb, the amount of FcγRIIb bound per unit amount of antibody was calculated by correcting the amount of FcγRIIb bound determined from the sensorgram obtained by measurement with the amount of antibody captured on the chip surface. For binding measurement to FcRn, 50 mM Na-Phosphate, 150 mM NaCl, 0.05% Tween20 (pH 6.0) was used as a running buffer, and the dissociation constant KD (mol/L) was calculated using a steady state model (Table 28 ). Note that among the dissociation constants for FcγRIIIa in Table 28, the values marked with "*" are values calculated using the Steady state affinity model.

Binding analysis of Fc region variants to human FcγR and FcRn

All of the heterodimeric antibodies produced here had enhanced binding activity to FcγRIIa and FcγRIIIa compared to 04H1389-G1m/04L1615-k0MT. In addition, modifications to improve the blood dynamics of antibodies were introduced. H1389- Both Km473/04L1615-k0MT//04H1389-Hm482/04L1615-k0MT and 04H1389-Km481/04L1615-k0MT//04H1389-Hm498/04L1615-k0MT showed higher binding to human FcRn than the parent antibody before modification. The activity was enhanced, and the binding profile for FcγR binding activity was equivalent to that of the parent antibody.

Furthermore, 04H1389-Hm441 (SEQ ID NO: 136) has 04H1389 (SEQ ID NO: 136) as a heavy chain variable region, the same modification as HT441 was introduced into the heavy chain CH2 region, and Y349C/T366W was used as a heterodimerization modification in the CH3 region ( A gene with SEQ ID NO: 273) was created. In contrast, 04H1389-Hm451 was created in which N434A/Y436T/Q438R/S440E, which is a combination of modifications that enhance binding to human FcRn under acidic conditions and modifications that reduce binding to Rheumatoid factor, was introduced (sequence Number: 274). The amino acid sequences of antibody heavy chains Hm441 and Hm451 are shown in SEQ ID NOs: 277 and 278, respectively. A heterodimeric antibody was produced using 04H1389-Km473, 04H1389-Hm451, or 04H1389-Hm482 as the antibody heavy chain and 04L1305-k0MT as the antibody light chain. Table 29 shows the results of interaction analysis between the produced antibodies and human FcRn and human FcγR.

Binding analysis of Fc region variants to human FcγR and FcRn

The heterodimeric antibodies 04H1389-Km473/04L1305-k0MT//04H1389-Hm451/04L1305-k0MT and 04H1389-Km473/04L1305-k0MT//04H1389-Hm482/04L1305-k0MT are both naturally occurring type human IgG1 It was shown that binding to FcγRIIa and FcγRIIIa, which are active FcγRs, was enhanced compared to 04H1389-G1m/04L1305-k0MT, which has a constant region. Furthermore, all of these antibodies were shown to have enhanced binding ability to human FcRn compared to 04H1389-G1m/04L1305-k0MT.

Next, we compared the constant region variant found here that enhanced binding to FcγR with existing FcγR binding-enhancing variants. Antibody heavy chain MDX10D1H-Kn125 (SEQ ID NO: 217), MDX10D1H-Hl076 (SEQ ID NO: 218) having MDX10D1H (SEQ ID NO: 154) as the heavy chain variable region and the heavy chain constant region listed in Table 26 , MDX10D1H-Kn462 (SEQ ID NO: 219), MDX10D1H-Hl445 (SEQ ID NO: 220), MDX10D1H-Kn461 (SEQ ID NO: 221), MDX10D1H-Hl443 (SEQ ID NO: 222), and the CH2 region of native human IgG1. A gene of MDX10D1H-G1m (SEQ ID NO: 210) was created. Antibody heavy chain MDX10D1H-GASDIE (SEQ ID NO:: 215) gene was created. In addition, the antibody heavy chain MDX10D1H-GASDALIE ( The gene with SEQ ID NO: 216) was created. MDX10D1L-k0MT (SEQ ID NO: 211) was used as the antibody light chain to produce the desired antibody. The binding activity to these human FcγRs was measured by the method using the aforementioned CaptureSelect Human Fab-kappa Kinetics Biotin Conjugate (Table 30).

Binding analysis of Fc region variants to human FcγR

The value of "KD (M) for hFcγR" in the table indicates the dissociation constant for each listed FcγR, and "binding amount" is the amount of FcγRIIb per unit antibody amount when interacting with FcγRIIb at 1000 nM. Indicates the amount of binding. "Relative value for KD of G1m and hFcγRs" indicates the value obtained by dividing the KD value of MDX10D1H-G1m/MDX10D1L-k0MT for each FcγR by the KD value of each variant, and "Relative binding amount" indicates the value for each variant for FcγRIIb. The value obtained by dividing the binding amount of MDX10D1H-G1m/MDX10D1L-k0MT by the binding amount of MDX10D1H-G1m/MDX10D1L-k0MT is shown.
The prepared heterodimers, MDX10D1H-Kn125/MDX10D1H-Hl076/MDX10D1L-k0MT, MDX10D1H-Kn462/MDX10D1H-Hl445/MDX10D1L-k0MT, MDX10D1H-Kn461/MDX10D1H-Hl443/MDX1 Both 0D1L-k0MT have existing FcγR binding The binding to FcγRIIIa was enhanced compared to the enhancing antibodies MDX10D1H-GASDIE/MDX10D1L-k0MT and MDX10D1H-GASDALIE/MDX10D1L-k0MT. It was also shown that MDX10D1H-Kn462/MDX10D1H-Hl445/MDX10D1L-k0MT has approximately two-fold enhanced binding to FcγRIIaH compared to MDX10D1H-GASDIE/MDX10D1L-k0MT, an existing FcγRIIa-enhancing antibody.

(6-2) Evaluation of in vitro ADCC activity of various antibodies with modified constant regions
hFcγRIIIaV ADCC Reporter Bioassay, Core Kit (Promega) was used for in vitro ADCC activity measurement. To each well of a 96-well plate, 25 μL of hCTLA4-CHO cells adjusted to a concentration of 2 × 10 ⁶ /mL with medium was added as target cells, and Assay Buffer (90% RPMI1640, 10% FBS) was added to the medium. used. Next, 25 μL of each antibody solution diluted with assay buffer to a final concentration of 0, 0.001, 0.01, 0.1, and 1 μg/mL was added, and finally, the effector cell solution was diluted to 6×10 ⁶ /mL with the medium. 25 μL of hFcγRIIIaV-expressing Jurkat cells (included with the kit) prepared in 2018 was added, mixed to a total of 75 μL, and left overnight at 37° C. in a 5% CO ₂ incubator. The plate was then left at room temperature for 15 minutes, and 75 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of a well to which no antibody was added was defined as fold induction, and was used as an index for evaluating ADCC of each antibody. The results obtained are shown in Figure 23. In the figure, fold induction is expressed as relative luminescence amount (RLU).

These results showed that the antibody with the modified Fc had a stronger ADCC activity against hCTLA4-CHO cells than that of the wild-type human IgG1 constant region.

(6-3) Evaluation of in vitro ADCP activity of various antibodies with modified constant regions
hFcγRIIaH ADCP Reporter Bioassay, Core Kit (Promega) was used for in vitro ADCC activity measurement. To each well of a 96-well plate, 25 μL of hCTLA4-CHO cells adjusted to a concentration of 1×10 ⁶ /mL with medium was added as target cells, and Assay Buffer (4% Low IgG serum in RPMI1640) was added to the medium. used. Next, 25 μL of each antibody solution diluted with assay buffer to a final concentration of 0, 0.001, 0.01, 0.1, and 1 μg/mL was added, and finally, the hFcγRIIaH-expressing Jurkat cells included in the kit were added as an effector cell solution. 25 μL was added and mixed for a total of 75 μL, and then left overnight at 37° C. in a 5% CO ₂ incubator. The hFcγRIIaH-expressing Jurkat cells had a cell fluid density of 8.25×10 ⁵ /mL. The plate was then allowed to stand at room temperature for 15 minutes, and 75 μl of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of a well to which no antibody was added was defined as fold induction, and was used as an index for evaluating the ADCP of each antibody. The results obtained are shown in Figure 24. In the figure, fold induction is expressed as relative luminescence amount (RLU).

These results showed that the antibody with the modified Fc had a stronger ADCP activity against hCTLA4-CHO cells than that of the wild-type human IgG1 constant region.

(6-4) Evaluation of in vitro ADCC activity of anti-CTLA4 switch antibody with modified Fc Name: SW1610-ART6, 04H1654-Kn462/04L1612-lam1//04H1656-Hl445/04L1612-lam1 Abbreviation: SW1612-ART6, and 04H1389-Kn462/04L1305-k0MT//04H1389-Hl445/04L130 5-k0MT Abbreviation: SW1389 -ART6) was created.
The SW1610-ART6 antibody has one heavy chain variable region 04H1654 (SEQ ID NO: 35) as a constant region, human heavy chain constant region Kn462 (SEQ ID NO: 43), and the other heavy chain variable region 04H1656 (SEQ ID NO: 37) as a constant region. ) is linked to the human heavy chain constant region Hl445 (SEQ ID NO: 44), and the light chain variable region 04L1610 (SEQ ID NO: 39) is linked to the wild type human light chain constant region lam1 (SEQ ID NO: 53). It was expressed and purified using methods known to those skilled in the art.
The SW1612-ART6 antibody has one heavy chain variable region 04H1654 (SEQ ID NO: 35) as a constant region, human heavy chain constant region Kn462 (SEQ ID NO: 43), and the other heavy chain variable region 04H1656 (SEQ ID NO: 37) as a constant region. ) is linked to the human heavy chain constant region Hl445 (SEQ ID NO: 44), and the light chain variable region 04L1612 (SEQ ID NO: 40) is linked to the wild type human light chain constant region lam1 (SEQ ID NO: 53). It was expressed and purified using methods known to those skilled in the art.
SW1389-ART6 antibody has two heavy chain variable regions 04H1389 (SEQ ID NO: 29) and human heavy chain constant region Kn462 (SEQ ID NO: 43) and human heavy chain constant region Hl445 (SEQ ID NO: 44) as constant regions, respectively. After ligation, the light chain variable region 04L1305 (SEQ ID NO: 30) was expressed and purified using the wild type human light chain constant region k0MT (SEQ ID NO: 33) by a method known to those skilled in the art.

hFcγRIIIaV ADCC Reporter Bioassay, Core Kit (Promega) was used for in vitro ADCC activity measurement. To each well of a 96-well plate, 12.5 μL of hCTLA4-CHO cells adjusted to a concentration of 2 × 10 ⁶ /mL with medium was added as target cells, and Assay Buffer (4% Low IgG Serum in RPMI1640) was added to the medium. used. Next, ATP solution diluted with assay buffer to a final concentration of 0, 100 μM, and SW1389-ART6 diluted with assay buffer to a final concentration of 0, 0.001, 0.01, 0.1, 1, 10 μg/mL. , SW1610-ART6 and SW1612-ART6 antibody solutions were added sequentially, and finally, 25 μL of hFcγRIIIaV-expressing Jurkat cells (included in the kit) adjusted to 3 × 10 ⁶ /mL with culture medium as an effector cell solution was added, making a total After mixing to a total volume of 75 μL, the mixture was left standing at 37° C. in a 5% CO ₂ incubator for 6 hours. The plate was then left at room temperature for 15 minutes, and 75 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of a well to which no antibody was added was defined as fold induction, and was used as an index for evaluating ADCC of each antibody. The obtained results are shown in Figure 25 (SW1389-ART6), Figure 26 (SW1610-ART6), and Figure 27 (SW1612-ART6). In the figure, fold induction is expressed as relative luminescence amount (RLU).

These results confirm that the ADCC activity of anti-CTLA4 switch antibodies with modified Fc against hCTLA4-CHO cells is different in the presence and absence of ATP, and that there is an ATP-dependent cytotoxic activity against hCTLA4-CHO cells. It was done.

(6-5) Evaluation of in vitro neutralizing activity of anti-CTLA4 switch antibodies Human antibodies having variable regions of SW1389, SW1610, SW1612, and SW1615 were created as modified anti-CTLA4 switch antibodies.
The SW1389 antibody uses 04H1389 (SEQ ID NO: 29) as the heavy chain variable region and 04L1305 (SEQ ID NO: 30) as the light chain variable region, which are linked to a human constant region, and then expressed by a method known to those skilled in the art. Refined.
The SW1610 antibody uses 04H1654 (SEQ ID NO: 35) and 04H1656 (SEQ ID NO: 37) as the heavy chain variable region and 04L1610 (SEQ ID NO: 39) as the light chain variable region, which are linked to a human constant region. It was expressed and purified by methods known to those skilled in the art.
The SW1612 antibody uses 04H1654 (SEQ ID NO: 35) and 04H1656 (SEQ ID NO: 37) as the heavy chain variable region and 04L1612 (SEQ ID NO: 40) as the light chain variable region, which are linked to a human constant region. It was expressed and purified by methods known to those skilled in the art.
The SW1615 antibody uses 04H1389 (SEQ ID NO: 29) as the heavy chain variable region and 04L1615 (SEQ ID NO: 34) as the light chain variable region, which are linked to a human constant region, and then expressed by a method known to those skilled in the art. Refined.
CTLA-4 Blockade Bioassay (Promega) was used for in vitro neutralization activity measurement. To each well of a 96-well plate, 25 μL of aAPC-Raji cells included in the kit, whose concentration was adjusted to 1 × 10 ⁶ /mL with medium, were added as target cells, and Assay Buffer (10% FBS in RPMI1640) was added to the medium. used. Next, ATP solution diluted with assay buffer to a final concentration of 0, 100 μM, SW1389, SW1610 diluted with assay buffer to a final concentration of 0, 0.001, 0.01, 0.1, 1, 10 μg/mL , Antibody solutions containing the variable regions of SW1612 and SW1615 were added sequentially, and finally, as an effector cell solution, 25 μL of IL2-luc2-CTLA4-Jurkat cells (included in the kit) adjusted to 2 × 10 ⁶ /mL with culture medium. After the mixture was added and mixed to a total of 75 μL, it was left standing at 37° C. for 6 hours in a 5% CO ₂ incubator. The plate was then left at room temperature for 15 minutes, and 75 μL of Bio-Glo reagent was added to each well. The Bio-glo Luciferase Assay System (Buffer and Substrate) was used as the Bio-Glo reagent. The luminescence of each well was then measured using a plate reader. The value obtained by dividing the luminescence value of each well by the luminescence value of a well to which no antibody was added was defined as fold induction, and was used as an index for evaluating the neutralizing activity of each antibody. The obtained results are shown in Figure 28 (SW1389), Figure 29 (SW1610), Figure 30 (SW1612), and Figure 31 (SW1615). In the figure, fold induction is expressed as relative luminescence amount (RLU).

From this result, it was confirmed that the neutralizing activity of the anti-CTLA4 switch antibody against hCTLA4-expressing cells was different in the presence and absence of ATP, and that ATP-dependent neutralizing activity existed.

(6-6) In vitro cytotoxic activity evaluation of anti-CTLA4 switch antibody against CTLA4-positive regulatory T cells Abbreviation: SW1610-ART5+ACT1, 04H1654-KT473/04L1610-lam1//04H1656-HT482/04L1610-lam1 Abbreviation: SW1610-ART6+ACT1, 04H1389-Km473/04L1305-k0MT//04H1389-Hm451/ 04L1305-k0MT Abbreviation: SW1389-ART5+ACT1), 04H1389-Km473/04L1305-k0MT//04H1389-Hm482/04L1305-k0MT Abbreviation: SW1389-ART6+ACT1) were produced.
The SW1610-ART5+ACT1 antibody has 04H1654-KT473 (SEQ ID NO: 184) as one heavy chain, 04H1656-HT451 (SEQ ID NO: 272) as the other heavy chain, and 04L1610-lam1 (SEQ ID NO: 272) as the light chain. 161), and was expressed and purified by a method known to those skilled in the art.
The SW1610-ART6+ACT1 antibody has 04H1654-KT473 (SEQ ID NO: 184) as one heavy chain, 04H1656-HT482 (SEQ ID NO: 185) as the other heavy chain, and 04L1610-lam1 (SEQ ID NO: 185) as the light chain. 161), and was expressed and purified by a method known to those skilled in the art.
The SW1389-ART5+ACT1 antibody has 04H1389-Km473 (SEQ ID NO: 203) as one heavy chain, 04H1389-Hm451 (SEQ ID NO: 274) as the other heavy chain, and 04L1305-k0MT (SEQ ID NO: 274) as the light chain. 275), and was expressed and purified by a method known to those skilled in the art.
The SW1389-ART6+ACT1 antibody has 04H1389-Km473 (SEQ ID NO: 203) as one heavy chain, 04H1389-Hm482 (SEQ ID NO: 204) as the other heavy chain, and 04L1305-k0MT (SEQ ID NO: 204) as the light chain. 275), and was expressed and purified by a method known to those skilled in the art.

The in vitro cytotoxic activity of the prepared anti-CTLA4 switch antibody having the modified Fc against CTLA4-positive regulatory T cells (CD3 ⁺ CD4 ⁺ CD25 ⁺ CD45RA ^- CTLA4 ⁺ ) was evaluated. First, human PBMC (CTL Cryopreserved Human PBMC, CTL) were frozen and thawed to a cell density of 2 × 10 ⁶ cells/mL in 50 U/ml interleukin-2 (IL-2)/RPMI/10% FBS. The cells were suspended and cultured at 37° C. for 4 days in a 5% CO ₂ incubator. After 4 days, cells were harvested and washed twice with RPMI/10% FBS, then seeded at 100 μL into each well of a 96-well U-bottom plate (8 x 10 ⁵ cells/well or 5 x 10 ⁵ cells). 25 μL of KLH-G1m solution adjusted to 8 mg/ml in RPMI/10% FBS was added to each well, followed by each concentration (0, 2.4, 8, 24, 80 μg/mL, 0, 0.8, 8, 80, 800 μg/mL) of an antibody solution prepared in RPMI/10% FBS was added at 25 μL into each well of a 96-well U-bottom plate. Furthermore, 50 μL of ATP solution adjusted to 0 or 400 μM with RPMI/10% FBS was added, and after being well suspended, it was left standing at 37 °C in a CO ₂ incubator for 6 hours (every 2 hours). 5 μL of ATP solution adjusted to 0 or 4000 μM was added twice in total). After 6 hours, PBMC were collected and washed twice with auto MACS Rinsing Solution (Milteny), then reacted with the following antibodies, and the fraction of immune cells present was analyzed by FACS analysis. Viability determination reagents (Biolegend, Zombie Aqua), anti-CD3 antibody (BD, clone: UCHT1), anti-CD4 antibody (BD, clone: RPA-T4), anti-CD8 antibody (BD, clone: SK1), anti-CD45RA antibody ( Biolegend, clone: HI100), anti-CD25 antibody (BD, clone: 2A3), anti-CD16 antibody (Biolegend, clone: 3G8), anti-CD56 antibody (Biolegend, clone: HCD56), anti-CTLA4 antibody (Biolegend, clone: BNI3). FACS analysis was performed on a BD LSR Fortessa The relative value was defined as the survival rate (%) of CTLA4-positive regulatory T cells, and was used as an index when evaluating the cytotoxic activity of each antibody.The obtained results are shown in Figure 32 (SW1389-ART5+ACT1). , shown in Figure 33 (SW1389-ART6+ACT1), Figure 34 (SW1610-ART5+ACT1) and Figure 35 (SW1610-ART6+ACT1).
These results show that the cytotoxic activity of anti-CTLA4 switch antibodies with modified Fc against CTLA4-positive regulatory T cells is different in the presence and absence of ATP, and the cytotoxic activity against CTLA4-positive regulatory T cells that is ATP-dependent is different in the presence and absence of ATP. It has been confirmed that it exists.

Although the foregoing invention has been described in detail using examples and illustrations for purposes of clarity of understanding, the description and illustrations herein should not be construed as limiting the scope of the invention. do not have. The disclosures of all patent and scientific publications cited herein are expressly incorporated by reference in their entirety.

The anti-CTLA-4 antibodies of the present disclosure and methods of using them have immune cell activating activity, cytotoxic activity, and/or antitumor activity, while having a low effect on non-tumor tissues such as normal tissues. It can be used in the development, manufacture, provision, use, etc. of medicines with few side effects. Furthermore, the polypeptide containing a mutant Fc region of the present disclosure and its production and use methods can be used in the development, production, provision, use, etc. of such pharmaceuticals.

Claims (7)

(a) HVR-H1 (SEQ ID NO: 223) comprising the amino acid sequence SX ₁ TMN, where X ₁ is H, A, R, or K;
(b) contains the amino acid sequence SISX ₁ X ₂ SX ₃ YIYYAX ₄ SVX ₅ G, where X ₁ is S _or T, X ₂ is R or Q, X ₃ is G or H, HVR-H2 (SEQ ID NO: 224), where X ₅ is K or R;
(c) HVR-H3 (SEQ ID NO: 225) comprising the amino acid sequence YGX ₁ REDMLWVFDY, where X ₁ is K or A;
(d) contains the amino acid sequence X ₁ GX ₂ STX ₃ VGDYX ₄ X ₅ VX ₆ , where X ₁ is T, D, Q, or E; X ₂ is T or P; X ₃ is D _or G; or T, X ₅ is Y or W, X ₆ is S or H HVR-L1 (SEQ ID NO: 226),
(e) comprises the amino acid sequence X ₁ TX ₂ X ₃ KPX ₄ , where X ₁ is E, F, or Y, X ₂ is S or I, _{X 3} is K or S, and HVR-L2 (SEQ ID NO: 227), and
(f) HVR-L3 comprising the amino acid sequence X ₁ TYAAPLGPX ₂ , where X ₁ is S or Q and X ₂ is M or T (SEQ ID NO: 228)
Anti-CTLA-4 antibodies, including: (1) (a) SEQ ID NO: HVR-H1 containing the 100 amino acid sequence;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 101;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 113;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 114;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 115;
(2) (a) SEQ ID NO: HVR-H1 containing the 100 amino acid sequence;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 104;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 116;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 115;
(3) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 105;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 106;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133;
(4) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 108;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 121;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 123;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 153;
(5) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 110;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 122;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133;
(6) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 112;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133;
(7) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 111;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 152;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133;
(8) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 112;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133;
(9) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 111;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 152;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117;
(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133, or
(10) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107;
(b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 109;
(c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102;
(d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 130;
(e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117;
(f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133;
The antibody according to claim 1, comprising : (a) a VH sequence having at least 95% sequence identity with any one amino acid sequence of SEQ ID NOs: 83-86, 98, 135-141;
(b) a VL sequence having at least 95% sequence identity with any one amino acid sequence of SEQ ID NOs: 88-95, 97, 99, 134, 144-149; or
(c) A VH sequence having an amino acid sequence of any one of SEQ ID NOs: 83-86, 98, 135-141, and an amino acid sequence of any one of SEQ ID NOs: 88-95, 97, 99, 134, 144-149 2. The antibody of claim 1, comprising a VL sequence. (1) VH sequence with sequence number: 98 and VL sequence with sequence number: 99 ,
( 2 ) VH sequence with SEQ ID NO: 83 and VL sequence with SEQ ID NO: 97 ,
( 3 ) VH sequence with sequence number: 86 and VL sequence with sequence number: 134 ,
( 4 ) VH sequence with SEQ ID NO: 136 and VL sequence with SEQ ID NO: 95 ,
( 5 ) VH sequence with SEQ ID NO: 140 and VL sequence with SEQ ID NO: 146,
( 6 ) VH sequence with SEQ ID NO: 141 and VL sequence with SEQ ID NO: 146,
( 7 ) VH sequence with SEQ ID NO: 140 and VL sequence with SEQ ID NO: 147,
( 8 ) VH sequence with SEQ ID NO: 141 and VL sequence with SEQ ID NO: 147 ,
( 9 ) VH sequence with SEQ ID NO: 136 and VL sequence with SEQ ID NO: 149,
( 10 ) A first variable region comprising the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 146, and a second variable region comprising the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 146, or
( 11 ) a first variable region comprising the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 147, and a second variable region comprising the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 147,
Anti-CTLA-4 antibodies, including: 5. A pharmaceutical formulation comprising an antibody according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. 6. A pharmaceutical formulation according to claim 5 for use in the treatment of tumors. 7. The pharmaceutical formulation according to claim 6 , wherein immunity is activated at a lower dose in tumor tissue compared to non-tumor tissue .

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